Steroid Hormone and Luteinizing Hormone Concentrations in the Anestrous Sow Glen W. Almond and Gary D. Dial
ABSTRACT
RESUME
This investigation characterized serum concentrations of luteinizing hormone (LH), estradiol-17j (E2), progesterone (P4) and cortisol (C) in anestrous sows. Twenty-two sows that had not returned to estrus within 45
Cette etude presente les concentrations seriques de l'hormone luteinisante (LH), de l'estradiol-17,B (E2), de la progesterone (P4) et du cortisol (C) chez des truies en anoestrus. Une canule fut installee non chirurgicalement dans la veine jugulaire chez vingt-deux truies qui n'avaient pas presente un oestrus en dedans de 45 jours apres le sevrage (truies anoestrales) et chez 10 truies ayant presente un oestrus en dedans de sept jours suivant le sevrage. Des echantillons de sang furent prelev's a l'intervalles de 6 h pendant sept jours et a intervalles de 15 minutes pendant 8 h au 5e jour apres la canulation. Les concentrations de la LH serique furent determinees chez tous les echantillons tandis que les niveaux de C, E2 et P4 furent determines des serums recoltes a intervalles de 6 h. Les concentrations seriques de la P4 chez les truies en anoestrus furent constamment inferieures a 0.5 ng/mL et les niveaux de E2 s'etendaient de 10 a 19 pg/mL. Les concentrations de la LH demeurerent inferieures a 1.0 ng/mL chez les truies en anoestrus tandis qu'un pic de LH preovulatoire fut observe chez cinq des dix truies demontrant un cycle oestral. Il y avait un rythme circadien de la moyenne des niveaux de C avec des pics se produisant a 0600 ou 2400 h tandis que les niveaux les plus bas etaient observes a 12004 et 1800 h. On a note peu de difference des taux de C entre les truies anoestrales et
days after weaning (anestrous sows), and ten sows that had returned to estrus within seven days following weaning (cyclic sows) were nonsurgically fitted with indwelling jugular vein cannulae. Blood samples were collected at 6 h intervals for seven days and at 15 min intervals for 8 h on the flfth day after cannulation. Serum LH concentrations were determined in all samples, while C, E2 and P4 levels were quantitated in serum collected at 6 h intervals. Serum P4 concentrations in anestrous sows were consistently less than 0.5 ng/mL, and E2 levels ranged from 10 to 19 pg/mL. Concentrations of LH remained less than 1.0 ng/mL in anestrous sows, whereas a preovulatory LH surge was observed in five of ten cyclic sows. There was a circadian rhythm in mean C levels with C peaks occurring at 0600 or 2400 h and nadir levels observed at 1200 and 1800 h. Few differences in C levels were detected between anestrous and cyclic sows. It was evident that anestrous sows did not exhibit cyclic or predictable variations in steroid hormone concentrations. Unfortunately, the results of this study failed to elucidate the endocrine pathogenesis of the anestrous sow.
cycliques. Il est evident que les truies anoestrales ne montrerent pas de variations cycliques ou previsibles des concentrations des hormones steroidiennes. Malheureusement les resultats de cette etude ne permettent pas d'elucider la pathogenie endocrinienne de l'anoestrus chez la truie.
INTRODUCTION Nearly all sows return to estrus within 3 to 14 days following weaning during most seasons of the year (1-3). There is an increase in the mean interval from weaning to reservice and an increased prevalence of sows, especially primiparous sows, failing to return to estrus following weaning during the summer and early autumn months (4-6). Following weaning, an increase in the number of luteinizing hormone (LH) pulses occurs in sows destined to return to estrus (7). There is a corresponding increase in the production of ovarian estrogens as follicle size increases (8,9). Serum changes in LH following weaning reflect increases in hypothalamic content of gonadotropin-releasing hormone (GnRH) and anterior pituitary concentrations of LH (10). In contrast to the sow that returns to estrus following weaning, dynamic changes in LH secretion and estradiol-17/3 (E2) concentrations do not occur during postweaning in sows failing to return to estrus (1 1,12).
Department of Food Animal and Equine Medicine, North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606. Reprint requests to Dr. G.D. Dial, Department of Large Animal Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108. This research was supported by USDA Special Grant 84-CRSR-2-2416, USDA Animal Health formula ends and the North Carolina Veterinary Competitive Grants Program. Submitted July 21, 1989.
Can J Vet Res 1990; 54: 209-214
209
In an effort to better understand anestrus in swine, this investigation characterized E2, LH and progesterone (P4) concentrations in sows that had remained persistently anestrous following weaning. Changes in hormone concentrations in diestrous and periestrous sows were determined as reference for the anestrous sow. Since the adrenal gland has been suggested to be involved in the inhibition of ovulation and LH secretion in swine (13-15), changes in serum cortisol (C) concentrations were also evaluated. 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. Thirty-two crossbred, primiparous sows were obtained from a commercial farm and transported to research facilities at North Carolina State University during July to October, 1986. Sows (n = 22) had either failed to exhibit estrus within 45 days after weaning and were classified as anestrous, or had returned to estrus within seven days following weaning and were classified as cyclic (n = 10). Animals were nonsurgically fitted upon arrival (day 1) with indwelling jugular vein cannulae. Cannulae were inserted through a 12 ga needle under local anesthesia and were taped to the skin with elastic tape. Blood samples were collected at 15 min intervals for 8 h on day 5. Sows were ovariectomized after seven days of blood collection at 6 h intervals. Examination of ovaries of anestrous sows revealed an absence of corpora lutea, follicles less than 5 mm in diameter, and well-regressed corpora albicantia. Cyclic sows were classified as either diestrous (n = 5) or periestrous (n = 5), based on ovarian structures observed at ovariectomy, serum hormone profiles prior to ovariectomy, and signs of estrus observed during twice daily boar exposure. Sera were frozen at -20°C until assayed for hormone concentrations. Luteinizing hormone concentrations were determined in all serum samples, and C, E2 and P4 levels were quantitated in sera collected at 6 h intervals. 210
ANALYSIS OF HORMONE CONCENTRATIONS
Serum LH concentrations were determined using a previously validated double antibody radioimmunoassay (RIA) (16). Niswender's No. 566 antiporcine LH serum was used in a final dilution of 1:32,000. A reaction of purified ovine LH preparation LER 1056-C2 with '25iodine in the presence of chloramine-T was used to radioiodinate LH. Each serum sample was assayed in duplicate aliquots of 200 ,uL. Results are expressed in terms of purified porcine reference standard LER 786-3, which had a biological activity of 0.65 NIH-LH-SIU/mg. The intra-assay coefficients of variation (CV) for both low (1.75 ng/ mL) and high (9.5 ng/mL) LH reference sera were consistently 95%. The CV of replicate measurements of samples were consistently < 5%. The intra-assay CV for the E2 RIA (n = 10 assays) for the low (25 pg/ mL) and high (165 pg/ mL) reference sera were < 5%. The interassay CV were 4.7% and 7.0% for the high and low sera, respectively. Sensitivity of the assay was approximately 1.0 pg/mL. Serum P4 concentrations were determined by a single-antibody, charcoal-dextran RIA using Gordon-
Sherwood antiserum No. 253 in a final dilution of 1:300,000 (16). Serum samples were reassayed if the standard error of duplicate measurements was > 10%. The interassay CV for the measurement of P4 in three samples of serum included in each assay were 8.1% (8 ng/ mL), 7.0% (16 ng/ mL) and 5.5% (32 ng/mL), respectively. The intra-assay CV for the reference sera was consistently < 5%. The sensitivity of the RIA (n = 7 assays), based on varying weights of P4 added to serum from a long-term ovariectomized sow, was approximately 0.25 ng/ mL. Cortisol concentrations were determined by solid phase '25iodine RIA using a commercial assay kit (Diagnostic Products Corporation, Los Angeles, California) validated for porcine serum. Antibody was immobilized on the wall of polypropylene tubes in a solid phase RIA. Neither extraction nor predilution was required. In brief, the assay technique required addition of 25 ,L of sample serum and 1.0 mL of buffered 1251cortisol to antibody-coated tubes. After a 45 min incubation in a 370C water bath, the supernatant was decanted, and the tube counted in a gamma counter. The samples were reassayed if the CV for duplicate serum aliquots exceeded 5%. Cortisol concentrations of 1, 5, 10, 20 and 50 ng/mL were used for the standard curve. There was no indication of lack of parallelism between serial dilutions of 32, 12, 4 and 2 ng/ mL preparations of porcine serum. The recovery of C (32, 12, 4 and 2 ng/ mL) added to 1 mL pig serum was 95 ± 2.3% (n = 10 observations). Cross-reactions of the C antibody to other endogenous steroids were
ABSTRACT
RESUME
This investigation characterized serum concentrations of luteinizing hormone (LH), estradiol-17j (E2), progesterone (P4) and cortisol (C) in anestrous sows. Twenty-two sows that had not returned to estrus within 45
Cette etude presente les concentrations seriques de l'hormone luteinisante (LH), de l'estradiol-17,B (E2), de la progesterone (P4) et du cortisol (C) chez des truies en anoestrus. Une canule fut installee non chirurgicalement dans la veine jugulaire chez vingt-deux truies qui n'avaient pas presente un oestrus en dedans de 45 jours apres le sevrage (truies anoestrales) et chez 10 truies ayant presente un oestrus en dedans de sept jours suivant le sevrage. Des echantillons de sang furent prelev's a l'intervalles de 6 h pendant sept jours et a intervalles de 15 minutes pendant 8 h au 5e jour apres la canulation. Les concentrations de la LH serique furent determinees chez tous les echantillons tandis que les niveaux de C, E2 et P4 furent determines des serums recoltes a intervalles de 6 h. Les concentrations seriques de la P4 chez les truies en anoestrus furent constamment inferieures a 0.5 ng/mL et les niveaux de E2 s'etendaient de 10 a 19 pg/mL. Les concentrations de la LH demeurerent inferieures a 1.0 ng/mL chez les truies en anoestrus tandis qu'un pic de LH preovulatoire fut observe chez cinq des dix truies demontrant un cycle oestral. Il y avait un rythme circadien de la moyenne des niveaux de C avec des pics se produisant a 0600 ou 2400 h tandis que les niveaux les plus bas etaient observes a 12004 et 1800 h. On a note peu de difference des taux de C entre les truies anoestrales et
days after weaning (anestrous sows), and ten sows that had returned to estrus within seven days following weaning (cyclic sows) were nonsurgically fitted with indwelling jugular vein cannulae. Blood samples were collected at 6 h intervals for seven days and at 15 min intervals for 8 h on the flfth day after cannulation. Serum LH concentrations were determined in all samples, while C, E2 and P4 levels were quantitated in serum collected at 6 h intervals. Serum P4 concentrations in anestrous sows were consistently less than 0.5 ng/mL, and E2 levels ranged from 10 to 19 pg/mL. Concentrations of LH remained less than 1.0 ng/mL in anestrous sows, whereas a preovulatory LH surge was observed in five of ten cyclic sows. There was a circadian rhythm in mean C levels with C peaks occurring at 0600 or 2400 h and nadir levels observed at 1200 and 1800 h. Few differences in C levels were detected between anestrous and cyclic sows. It was evident that anestrous sows did not exhibit cyclic or predictable variations in steroid hormone concentrations. Unfortunately, the results of this study failed to elucidate the endocrine pathogenesis of the anestrous sow.
cycliques. Il est evident que les truies anoestrales ne montrerent pas de variations cycliques ou previsibles des concentrations des hormones steroidiennes. Malheureusement les resultats de cette etude ne permettent pas d'elucider la pathogenie endocrinienne de l'anoestrus chez la truie.
INTRODUCTION Nearly all sows return to estrus within 3 to 14 days following weaning during most seasons of the year (1-3). There is an increase in the mean interval from weaning to reservice and an increased prevalence of sows, especially primiparous sows, failing to return to estrus following weaning during the summer and early autumn months (4-6). Following weaning, an increase in the number of luteinizing hormone (LH) pulses occurs in sows destined to return to estrus (7). There is a corresponding increase in the production of ovarian estrogens as follicle size increases (8,9). Serum changes in LH following weaning reflect increases in hypothalamic content of gonadotropin-releasing hormone (GnRH) and anterior pituitary concentrations of LH (10). In contrast to the sow that returns to estrus following weaning, dynamic changes in LH secretion and estradiol-17/3 (E2) concentrations do not occur during postweaning in sows failing to return to estrus (1 1,12).
Department of Food Animal and Equine Medicine, North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606. Reprint requests to Dr. G.D. Dial, Department of Large Animal Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108. This research was supported by USDA Special Grant 84-CRSR-2-2416, USDA Animal Health formula ends and the North Carolina Veterinary Competitive Grants Program. Submitted July 21, 1989.
Can J Vet Res 1990; 54: 209-214
209
In an effort to better understand anestrus in swine, this investigation characterized E2, LH and progesterone (P4) concentrations in sows that had remained persistently anestrous following weaning. Changes in hormone concentrations in diestrous and periestrous sows were determined as reference for the anestrous sow. Since the adrenal gland has been suggested to be involved in the inhibition of ovulation and LH secretion in swine (13-15), changes in serum cortisol (C) concentrations were also evaluated. 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. Thirty-two crossbred, primiparous sows were obtained from a commercial farm and transported to research facilities at North Carolina State University during July to October, 1986. Sows (n = 22) had either failed to exhibit estrus within 45 days after weaning and were classified as anestrous, or had returned to estrus within seven days following weaning and were classified as cyclic (n = 10). Animals were nonsurgically fitted upon arrival (day 1) with indwelling jugular vein cannulae. Cannulae were inserted through a 12 ga needle under local anesthesia and were taped to the skin with elastic tape. Blood samples were collected at 15 min intervals for 8 h on day 5. Sows were ovariectomized after seven days of blood collection at 6 h intervals. Examination of ovaries of anestrous sows revealed an absence of corpora lutea, follicles less than 5 mm in diameter, and well-regressed corpora albicantia. Cyclic sows were classified as either diestrous (n = 5) or periestrous (n = 5), based on ovarian structures observed at ovariectomy, serum hormone profiles prior to ovariectomy, and signs of estrus observed during twice daily boar exposure. Sera were frozen at -20°C until assayed for hormone concentrations. Luteinizing hormone concentrations were determined in all serum samples, and C, E2 and P4 levels were quantitated in sera collected at 6 h intervals. 210
ANALYSIS OF HORMONE CONCENTRATIONS
Serum LH concentrations were determined using a previously validated double antibody radioimmunoassay (RIA) (16). Niswender's No. 566 antiporcine LH serum was used in a final dilution of 1:32,000. A reaction of purified ovine LH preparation LER 1056-C2 with '25iodine in the presence of chloramine-T was used to radioiodinate LH. Each serum sample was assayed in duplicate aliquots of 200 ,uL. Results are expressed in terms of purified porcine reference standard LER 786-3, which had a biological activity of 0.65 NIH-LH-SIU/mg. The intra-assay coefficients of variation (CV) for both low (1.75 ng/ mL) and high (9.5 ng/mL) LH reference sera were consistently 95%. The CV of replicate measurements of samples were consistently < 5%. The intra-assay CV for the E2 RIA (n = 10 assays) for the low (25 pg/ mL) and high (165 pg/ mL) reference sera were < 5%. The interassay CV were 4.7% and 7.0% for the high and low sera, respectively. Sensitivity of the assay was approximately 1.0 pg/mL. Serum P4 concentrations were determined by a single-antibody, charcoal-dextran RIA using Gordon-
Sherwood antiserum No. 253 in a final dilution of 1:300,000 (16). Serum samples were reassayed if the standard error of duplicate measurements was > 10%. The interassay CV for the measurement of P4 in three samples of serum included in each assay were 8.1% (8 ng/ mL), 7.0% (16 ng/ mL) and 5.5% (32 ng/mL), respectively. The intra-assay CV for the reference sera was consistently < 5%. The sensitivity of the RIA (n = 7 assays), based on varying weights of P4 added to serum from a long-term ovariectomized sow, was approximately 0.25 ng/ mL. Cortisol concentrations were determined by solid phase '25iodine RIA using a commercial assay kit (Diagnostic Products Corporation, Los Angeles, California) validated for porcine serum. Antibody was immobilized on the wall of polypropylene tubes in a solid phase RIA. Neither extraction nor predilution was required. In brief, the assay technique required addition of 25 ,L of sample serum and 1.0 mL of buffered 1251cortisol to antibody-coated tubes. After a 45 min incubation in a 370C water bath, the supernatant was decanted, and the tube counted in a gamma counter. The samples were reassayed if the CV for duplicate serum aliquots exceeded 5%. Cortisol concentrations of 1, 5, 10, 20 and 50 ng/mL were used for the standard curve. There was no indication of lack of parallelism between serial dilutions of 32, 12, 4 and 2 ng/ mL preparations of porcine serum. The recovery of C (32, 12, 4 and 2 ng/ mL) added to 1 mL pig serum was 95 ± 2.3% (n = 10 observations). Cross-reactions of the C antibody to other endogenous steroids were
