Pubertal Development in the Male Pig: Effects of Treatment with a Long-acting Gonadotropin-releasing Hormone Agonist on Plasma Luteinizing Hormone, Follicle Stimulating Hormone and Testosterone V.L. Trudeau, J.C. Meijer, J.H.F. Erkens, D.F.M.
ABSTRACT The effects of a long-acting gonadotropin-releasing hormone (GnRH) agonist, [D-Trp6]-GnRH (GnRH-A) on developmental profiles of plasma luteinizing hormone (LH), follicle stimulation hormone (FSH) and testosterone (T), and pituitary responsiveness to exogenous GnRH were studied in male Dutch Landrace x Large White crossbred pigs from 1 to 30 wk of age. Group 1 control animals (control; n = 12) were injected subcutaneously in the neck with vehicle at 1 and 16 wk of age. Group 2 animals (early treatment; n = 10) were injected with 600 jig [D-Trp6]-GnRH at 1 wk and with vehicle at 16 wk. Group 3 animals (late treatment; n = 8) were injected with vehicle and 3 mg GnRH-A at 1 and 16 wk, respectively. Group 4 animals (early plus late treatment; n = 9) were injected at both 1 and 16 wk with GnRH-A. Blood was collected by brachiocephalic puncture at weekly or biweekly intervals, and through brachiocephalic cannulae, to determine longitudinal profies of LH, FSH and T, and plasma gonadotropin responses to intravenous injection of GnRH (0.1 itg/kg), respectively. In control animals, LH and FSH declined over the first 5 wk of postnatal life and peaked again at 10-14 wk. Levels of both hormones were basal from 18 to 30 wk. Plasma T was high in the first week, declined progressively over the next few weeks and remained low until 24 wk when a transient increment was noted. The LH and FSH responses to
van
de Wiel and C.J.G. Wensing
acute GnRH stimulation were similar at 7 and 14 wk and declined significantly at 23 wk of age. Treatment with GnRH-A at 1 wk but not 16 wk
resulted in significantly reduced LH, FSH and T levels for a period of 2 wk. Testosterone levels were lower at 24 wk in male pigs injected with GnRH-A at both 1 and 16 wk, suggesting that pubertal development was slightly delayed. Histological assessment of the testes revealed significant reductions in seminiferous tubule diameter in 30 wk old males treated with GnRH-A, indicating that GnRH agonist-induced suppression of pituitary-testicular function at 1 and/or 16 wk of age has important effects on development of seminiferous tubules in the male pig.
RESUME Les effets d'un agoniste a action prolongee de l'hormone de liberation des gonadotropines (GnRH-A), [D-Trp6] GnRH, sur les concentrations plasmatiques des hormones luteinisante (LH), folliculo-stimulante (FSH), et de la testosterone ont ete etudies chez des verrats (Landrace x "Large White" ages de 1 a 30 sem) ainsi que Ia reponse de l'adenohypophyse a une stimulation exogene A la GnRH. Les 12 animaux du premier groupe servirent de temoin et recurent un placebo a l'age de 1 et 16 semaines. Les 10 animaux du deuxieme groupe (traitement precoce) ne recurent le traitement avec l'agoniste (GnRH-A) qu'a la premiere semaine et le placebo
A l'age de 16 semaines. Les 8 animaux du troisieme groupe (traitement tardif) recurent le placebo a l'age de 1 semaine et le traitement a 16 semaines alors que les 9 animaux du quatrieme groupe (traitement precoce et tardif) recurent le traitement aux Ages de 1 et 16 semaines. Les profils seriques de LH, FSH et de testosterone ainsi que les concentrations d'hormones gonadotropes suivant l'administration intraveineuse de GnRH (0,1 mg/kg) furent evalues sur une base hebdomadaire ou bi-hebdomadaire. Les concentrations seriques de LH et FSH diminuerent durant les cinq premieres semaines de vie et furent suivies d'un pic entre l'age de 10 et de 14 semaines pour demeurer faibles et stables entre l'age de 18 et de 30 semaines. Les concentrations de testosterone plasmatique furent elevees durant la premiere semaine pour diminuer progressivement pendant les semaines suivantes et resterent faibles jusqu'a la 24ieme semaine de vie durant laquelle on nota une augmentation
passagere.
Une stimulation par la GnRH une relache des h-ormones gonadotropes (LH et FSH) similaire A 7 et 14 semaines pour diminuer significativement a l'age de 23 semaines. Le traitement par l'agoniste de la GnRH diminua les concentrations sfriques de LH, FSH et de testosterone pour une periode de 2 semaines seulement chez les verrats ages de 1 semaine. L'administration de cet agoniste a l'age de 1 et 16 semaines fit que la concentration serique en testosterone fut plus faible entraina
Department of Functional Morphology, Veterinary Faculty, University of Utrecht, The Netherlands (Trudeau, Meijer, Wensing) and Research Institute for Animal Production "Schoonoord", Zeist, The Netherlands (Erkens, van de Wiel). Present address of Dr. V.L. Trudeau: Department of Neuroscience, CSB Room 9, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario K1Y 4E9. Present address of Dr. J.C. Meijer: Trouw International BV, Putten, The Netherlands. Present address of Dr. C.J.G. Wensing: Central Institute for Veterinary Research, C.D.I., Lelystad, The Netherlands. Submitted August 19, 1991.
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a l'age de 24 semaines, ce qui suggere pituitary responsiveness to acute synthetic GnRH (Fertagyl, Intervet, un leger retard du developpement GnRH stimulation during maturation. Boxmeer, The Netherlands) in sterile pubertaire. Le diametre des tubules Since inhibition of gonadotropin and saline were examined at 7, 14 and seminiferes fut significativement red'uit sex steroid secretion by desensitization 23 wk. The brachiocephalic vein of chez les verrats a l'age de 30 semaines of the pituitary to GnRH can be four to six boars per treatment group qui resurent l'agoniste, ce qui souligne achieved by administration of long- was cannulated as previously reported son action negative sur I'axe ante- acting GnRH agonists (12,14,15), we (11). Two days following cannulation, hypophysio-testiculaire aussi bien a also investigated the effects of the five blood samples were taken at I'age de 1 que de 16 semaines. (Traduit administration of a slow release for- 30 min intervals before the injection of par Dr Guy-Pierre Martineau) mulation of the GnRH agonist [D- GnRH, thereafter samples were taken Trp6]-GnRH (GnRH-A) on profiles at 10, 20, 30, 40, 60, 90, 120 and of LH, FSH and T, and on gonadal 150 min. The mean of the three lowest maturation in the developing male pig. pre-GnRH injection values was conINTRODUCTION sidered to represent basal LH or FSH concentrations for individual pigs. Plasma gonadotropin levels have MATERIALS AND METHODS Mean values represent the mean of the been reported to be relatively stable five pre-GnRH values. The magnitude throughout pubertal development of EXPERIMENTAL GROUPS the domestic male pig by several Thirty-nine male Dutch Landrace x of a response was defined as the maxauthors (1,2), in marked contrast to Large White crossbred pigs born in imum hormone levels minus the basal our own work (3,4) which demon- mid-February and originating from concentration (16). strated that plasma luteinizing hor- 16 litters (two to four piglets from each At slaughter, testes, epididymes and mone (LH) and follicle stimulating litter) were allotted to four groups at accessory sex glands were dissected, hormone (FSH) levels varied con- 1 wk of age. Group 1 control animals measured, weighed and processed for siderably from birth to puberty. An (control; n = 12) were injected sub- histology. Fresh smears of deferent early postnatal period of high cir- cutaneously in the neck with a carboxy- duct contents were checked for the culating levels of LH, FSH and methyl cellulose/Tween 80 vehicle at presence of spermatozoa. testosterone (T) was separated from 1 and 16 wk of age. Group 2 animals the pubertal period of gonadotropic (early treatment; n = 10) were injected RADIOIMMUNOASSAYS (RIA) The LH and FSH concentrations and gonadal activity by a period of with 600 Ag [D-Trp6]-GnRH (dose relative quiescence (3-5). Furthermore, was approximately 200-400 yg per kg were estimated with validated RIA there are several reports (1,5-8) of metabolic weight; kg0 75) in micro- (17). The intra- and interassay coeffimaturational profiles of plasma T for capsules (Decapeptyl, Cytotech, cients of variation were less than 8%Wo the male pig which are in disagreement Switzerland; provided by Intervet, and less than 10% respectively for both as to the overall pattern of T secretion Boxmeer, The Netherlands) at 1 wk, assays. Minimum detection limits during development. Taking the pres- and with vehicle at 16 wk. Group 3 (defined as 95 % total binding) were ence of live spermatozoa in the animals (late treatment; n= 8) were 0.1 ng/mL for the LH-RIA and deferent ducts as a criterion, puberty injected with vehicle and 3 mg GnRH-A 0.3 ng/mL for the FSH-RIA. Testosterone was quantified in unexin male pigs is reached at about five (dose was approximately 200-300 jig months of age (6). per kg0_75 at 1 and 16 wk, respectively. tracted plasma using a solid-phase RIA Pituitary gonadotropin secretion is Group 4 animals (early plus late treat- (Coat-a-Count, Diagnostic Products primarily under the stimulatory control ment; n =9) were injected at both 1 Corporation, Los Angeles) as previof gonadotropin-releasing hormone and 16 wk with GnRH-A as described ously reported (11). The intra- and (GnRH) (9). Acute treatments with above. All piglets were left in their interassay coefficients of variation qo and the minimum GnRH stimulate pituitary gonado- respective litters until 5 wk of age, were less than 11% tropin release in the immature male pig when they were weaned, reallocated detection limit was 0.3 ng/mL. Cross(10,11) but it is not known if pituitary and regrouped into four large pens. reactivity of 5a-dihydrotestosterone is sensitivity to GnRH changes through- Animals were fed and maintained in approximately 8% in this assay. out pubertal development. Further- good health and were treated in accormore, the significance of the early dance with Canadian and Dutch Coun- HISTOLOGY Frozen sections of testes were postnatal increases in pituitary- cils on Animal Care Guidelines gonadal function (3,4,5) in pubertal throughout the experiment. The analyzed histochemically for activity of development in male pigs has not been groups were kept under identical con- 3,B-hydroxysteroid dehydrogenase (18). established. ditions until 30 wk of age when they Paraffin sections of testes were Given the inconsistent reports on were slaughtered. Blood was collected analyzed for differentiation of the developmental profiles of plasma by brachiocephalic venipuncture at seminiferous epithelium and for gonadotropin and T levels for the male weekly or biweekly intervals from 1 seminiferous tubular diameter as prepig, the first objective of the present until 28 wk of age. Plasma was kept viously reported (19,20). Briefly, one study was to reassess our previous at - 80°C until assayed for LH, FSH hundred randomly selected transverse sections of seminiferous tubules per work on sexual development in male and T. pigs (3,4,5,12,13) and to extend these Responses of plasma LH and FSH testis were classified on a scale of 1 to observations to include assessment of to intravenous injection of 0.1 "tg/kg 6 according to the most differentiated
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spermatogenic cell type present. A TABLE I. Plasma LH concentrations (ng/mL) from 1 to 28 wk of age in male pigs treated with at 1 wk (early treatment) and/or 16 wk (late treatment). Data are mean ± SEM. Number score of 1 represented tubules without GnRH-A of animals is indicated in parentheses spermatogenic cells, whereas a score of 6 represented tubules with elongated Treatment groups spermatogonia. A mean score was Week of age Control (12) Early (10) Late (8) Early plus late (9) calculated to obtain the spermatogenic 1 2.59± 0.49 2.78±0.46 2.30±0.22 2.93 ±0.97 index (19). 2 3.67 ±0.75 1.31 ± 0.75a 2.30±0.27 0.93 ± 0.43a DATA ANALYSIS
Hormonal data were statistically analyzed using the least squares method of analysis of variance (21). All data were fitted to a model that included the main effects of treatment and age. The treatment effect was tested using the boar-within-treatment mean square, while the effects of age and the treatment x age interaction were tested with the residual mean square. Data from maturational profiles and from GnRH responsiveness tests were used to calculate the ratio of plasma LH to FSH (LH/FSH ratio) as an indirect index of differential regulation of gonadotropin release. For GnRH response data the LH/FSH ratio was calculated for basal (preGnRH injection; mean of three lowest samples) and mean 1 h hormone increment (mean of five samples) following GnRH injection. One hour following GnRH injection was chosen since in the majority of animals, LH and FSH levels return to pre-injection levels within this period. Histological data were tested for differences between groups with the Kruskal-Wallis one factor analysis of variance. RESULTS HORMONE PROFILES
Maturational changes (age; p < 0.01) in mean LH concentrations were evident ([able I). In addition, GnRH-A treatment influenced LH levels (treatment; p < 0.05) and the maturational profile (age x treatment; p < 0.01). In the control group plasma LH levels were high in the first 3 wk and declined thereafter until 9 wk. Increased levels were noted in the 10-14 wk period and values declined thereafter. Early treatment with GnRH-A reduced LH levels for 2 wk following drug administration. Late treatment with GnRH-A was without effect. Although all groups had elevated LH concentrations at 10-16 wk, levels were particularly high in the control group at 104
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ABSTRACT The effects of a long-acting gonadotropin-releasing hormone (GnRH) agonist, [D-Trp6]-GnRH (GnRH-A) on developmental profiles of plasma luteinizing hormone (LH), follicle stimulation hormone (FSH) and testosterone (T), and pituitary responsiveness to exogenous GnRH were studied in male Dutch Landrace x Large White crossbred pigs from 1 to 30 wk of age. Group 1 control animals (control; n = 12) were injected subcutaneously in the neck with vehicle at 1 and 16 wk of age. Group 2 animals (early treatment; n = 10) were injected with 600 jig [D-Trp6]-GnRH at 1 wk and with vehicle at 16 wk. Group 3 animals (late treatment; n = 8) were injected with vehicle and 3 mg GnRH-A at 1 and 16 wk, respectively. Group 4 animals (early plus late treatment; n = 9) were injected at both 1 and 16 wk with GnRH-A. Blood was collected by brachiocephalic puncture at weekly or biweekly intervals, and through brachiocephalic cannulae, to determine longitudinal profies of LH, FSH and T, and plasma gonadotropin responses to intravenous injection of GnRH (0.1 itg/kg), respectively. In control animals, LH and FSH declined over the first 5 wk of postnatal life and peaked again at 10-14 wk. Levels of both hormones were basal from 18 to 30 wk. Plasma T was high in the first week, declined progressively over the next few weeks and remained low until 24 wk when a transient increment was noted. The LH and FSH responses to
van
de Wiel and C.J.G. Wensing
acute GnRH stimulation were similar at 7 and 14 wk and declined significantly at 23 wk of age. Treatment with GnRH-A at 1 wk but not 16 wk
resulted in significantly reduced LH, FSH and T levels for a period of 2 wk. Testosterone levels were lower at 24 wk in male pigs injected with GnRH-A at both 1 and 16 wk, suggesting that pubertal development was slightly delayed. Histological assessment of the testes revealed significant reductions in seminiferous tubule diameter in 30 wk old males treated with GnRH-A, indicating that GnRH agonist-induced suppression of pituitary-testicular function at 1 and/or 16 wk of age has important effects on development of seminiferous tubules in the male pig.
RESUME Les effets d'un agoniste a action prolongee de l'hormone de liberation des gonadotropines (GnRH-A), [D-Trp6] GnRH, sur les concentrations plasmatiques des hormones luteinisante (LH), folliculo-stimulante (FSH), et de la testosterone ont ete etudies chez des verrats (Landrace x "Large White" ages de 1 a 30 sem) ainsi que Ia reponse de l'adenohypophyse a une stimulation exogene A la GnRH. Les 12 animaux du premier groupe servirent de temoin et recurent un placebo a l'age de 1 et 16 semaines. Les 10 animaux du deuxieme groupe (traitement precoce) ne recurent le traitement avec l'agoniste (GnRH-A) qu'a la premiere semaine et le placebo
A l'age de 16 semaines. Les 8 animaux du troisieme groupe (traitement tardif) recurent le placebo a l'age de 1 semaine et le traitement a 16 semaines alors que les 9 animaux du quatrieme groupe (traitement precoce et tardif) recurent le traitement aux Ages de 1 et 16 semaines. Les profils seriques de LH, FSH et de testosterone ainsi que les concentrations d'hormones gonadotropes suivant l'administration intraveineuse de GnRH (0,1 mg/kg) furent evalues sur une base hebdomadaire ou bi-hebdomadaire. Les concentrations seriques de LH et FSH diminuerent durant les cinq premieres semaines de vie et furent suivies d'un pic entre l'age de 10 et de 14 semaines pour demeurer faibles et stables entre l'age de 18 et de 30 semaines. Les concentrations de testosterone plasmatique furent elevees durant la premiere semaine pour diminuer progressivement pendant les semaines suivantes et resterent faibles jusqu'a la 24ieme semaine de vie durant laquelle on nota une augmentation
passagere.
Une stimulation par la GnRH une relache des h-ormones gonadotropes (LH et FSH) similaire A 7 et 14 semaines pour diminuer significativement a l'age de 23 semaines. Le traitement par l'agoniste de la GnRH diminua les concentrations sfriques de LH, FSH et de testosterone pour une periode de 2 semaines seulement chez les verrats ages de 1 semaine. L'administration de cet agoniste a l'age de 1 et 16 semaines fit que la concentration serique en testosterone fut plus faible entraina
Department of Functional Morphology, Veterinary Faculty, University of Utrecht, The Netherlands (Trudeau, Meijer, Wensing) and Research Institute for Animal Production "Schoonoord", Zeist, The Netherlands (Erkens, van de Wiel). Present address of Dr. V.L. Trudeau: Department of Neuroscience, CSB Room 9, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario K1Y 4E9. Present address of Dr. J.C. Meijer: Trouw International BV, Putten, The Netherlands. Present address of Dr. C.J.G. Wensing: Central Institute for Veterinary Research, C.D.I., Lelystad, The Netherlands. Submitted August 19, 1991.
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Can J Vet Res 1992; 56: 102-109
a l'age de 24 semaines, ce qui suggere pituitary responsiveness to acute synthetic GnRH (Fertagyl, Intervet, un leger retard du developpement GnRH stimulation during maturation. Boxmeer, The Netherlands) in sterile pubertaire. Le diametre des tubules Since inhibition of gonadotropin and saline were examined at 7, 14 and seminiferes fut significativement red'uit sex steroid secretion by desensitization 23 wk. The brachiocephalic vein of chez les verrats a l'age de 30 semaines of the pituitary to GnRH can be four to six boars per treatment group qui resurent l'agoniste, ce qui souligne achieved by administration of long- was cannulated as previously reported son action negative sur I'axe ante- acting GnRH agonists (12,14,15), we (11). Two days following cannulation, hypophysio-testiculaire aussi bien a also investigated the effects of the five blood samples were taken at I'age de 1 que de 16 semaines. (Traduit administration of a slow release for- 30 min intervals before the injection of par Dr Guy-Pierre Martineau) mulation of the GnRH agonist [D- GnRH, thereafter samples were taken Trp6]-GnRH (GnRH-A) on profiles at 10, 20, 30, 40, 60, 90, 120 and of LH, FSH and T, and on gonadal 150 min. The mean of the three lowest maturation in the developing male pig. pre-GnRH injection values was conINTRODUCTION sidered to represent basal LH or FSH concentrations for individual pigs. Plasma gonadotropin levels have MATERIALS AND METHODS Mean values represent the mean of the been reported to be relatively stable five pre-GnRH values. The magnitude throughout pubertal development of EXPERIMENTAL GROUPS the domestic male pig by several Thirty-nine male Dutch Landrace x of a response was defined as the maxauthors (1,2), in marked contrast to Large White crossbred pigs born in imum hormone levels minus the basal our own work (3,4) which demon- mid-February and originating from concentration (16). strated that plasma luteinizing hor- 16 litters (two to four piglets from each At slaughter, testes, epididymes and mone (LH) and follicle stimulating litter) were allotted to four groups at accessory sex glands were dissected, hormone (FSH) levels varied con- 1 wk of age. Group 1 control animals measured, weighed and processed for siderably from birth to puberty. An (control; n = 12) were injected sub- histology. Fresh smears of deferent early postnatal period of high cir- cutaneously in the neck with a carboxy- duct contents were checked for the culating levels of LH, FSH and methyl cellulose/Tween 80 vehicle at presence of spermatozoa. testosterone (T) was separated from 1 and 16 wk of age. Group 2 animals the pubertal period of gonadotropic (early treatment; n = 10) were injected RADIOIMMUNOASSAYS (RIA) The LH and FSH concentrations and gonadal activity by a period of with 600 Ag [D-Trp6]-GnRH (dose relative quiescence (3-5). Furthermore, was approximately 200-400 yg per kg were estimated with validated RIA there are several reports (1,5-8) of metabolic weight; kg0 75) in micro- (17). The intra- and interassay coeffimaturational profiles of plasma T for capsules (Decapeptyl, Cytotech, cients of variation were less than 8%Wo the male pig which are in disagreement Switzerland; provided by Intervet, and less than 10% respectively for both as to the overall pattern of T secretion Boxmeer, The Netherlands) at 1 wk, assays. Minimum detection limits during development. Taking the pres- and with vehicle at 16 wk. Group 3 (defined as 95 % total binding) were ence of live spermatozoa in the animals (late treatment; n= 8) were 0.1 ng/mL for the LH-RIA and deferent ducts as a criterion, puberty injected with vehicle and 3 mg GnRH-A 0.3 ng/mL for the FSH-RIA. Testosterone was quantified in unexin male pigs is reached at about five (dose was approximately 200-300 jig months of age (6). per kg0_75 at 1 and 16 wk, respectively. tracted plasma using a solid-phase RIA Pituitary gonadotropin secretion is Group 4 animals (early plus late treat- (Coat-a-Count, Diagnostic Products primarily under the stimulatory control ment; n =9) were injected at both 1 Corporation, Los Angeles) as previof gonadotropin-releasing hormone and 16 wk with GnRH-A as described ously reported (11). The intra- and (GnRH) (9). Acute treatments with above. All piglets were left in their interassay coefficients of variation qo and the minimum GnRH stimulate pituitary gonado- respective litters until 5 wk of age, were less than 11% tropin release in the immature male pig when they were weaned, reallocated detection limit was 0.3 ng/mL. Cross(10,11) but it is not known if pituitary and regrouped into four large pens. reactivity of 5a-dihydrotestosterone is sensitivity to GnRH changes through- Animals were fed and maintained in approximately 8% in this assay. out pubertal development. Further- good health and were treated in accormore, the significance of the early dance with Canadian and Dutch Coun- HISTOLOGY Frozen sections of testes were postnatal increases in pituitary- cils on Animal Care Guidelines gonadal function (3,4,5) in pubertal throughout the experiment. The analyzed histochemically for activity of development in male pigs has not been groups were kept under identical con- 3,B-hydroxysteroid dehydrogenase (18). established. ditions until 30 wk of age when they Paraffin sections of testes were Given the inconsistent reports on were slaughtered. Blood was collected analyzed for differentiation of the developmental profiles of plasma by brachiocephalic venipuncture at seminiferous epithelium and for gonadotropin and T levels for the male weekly or biweekly intervals from 1 seminiferous tubular diameter as prepig, the first objective of the present until 28 wk of age. Plasma was kept viously reported (19,20). Briefly, one study was to reassess our previous at - 80°C until assayed for LH, FSH hundred randomly selected transverse sections of seminiferous tubules per work on sexual development in male and T. pigs (3,4,5,12,13) and to extend these Responses of plasma LH and FSH testis were classified on a scale of 1 to observations to include assessment of to intravenous injection of 0.1 "tg/kg 6 according to the most differentiated
103
spermatogenic cell type present. A TABLE I. Plasma LH concentrations (ng/mL) from 1 to 28 wk of age in male pigs treated with at 1 wk (early treatment) and/or 16 wk (late treatment). Data are mean ± SEM. Number score of 1 represented tubules without GnRH-A of animals is indicated in parentheses spermatogenic cells, whereas a score of 6 represented tubules with elongated Treatment groups spermatogonia. A mean score was Week of age Control (12) Early (10) Late (8) Early plus late (9) calculated to obtain the spermatogenic 1 2.59± 0.49 2.78±0.46 2.30±0.22 2.93 ±0.97 index (19). 2 3.67 ±0.75 1.31 ± 0.75a 2.30±0.27 0.93 ± 0.43a DATA ANALYSIS
Hormonal data were statistically analyzed using the least squares method of analysis of variance (21). All data were fitted to a model that included the main effects of treatment and age. The treatment effect was tested using the boar-within-treatment mean square, while the effects of age and the treatment x age interaction were tested with the residual mean square. Data from maturational profiles and from GnRH responsiveness tests were used to calculate the ratio of plasma LH to FSH (LH/FSH ratio) as an indirect index of differential regulation of gonadotropin release. For GnRH response data the LH/FSH ratio was calculated for basal (preGnRH injection; mean of three lowest samples) and mean 1 h hormone increment (mean of five samples) following GnRH injection. One hour following GnRH injection was chosen since in the majority of animals, LH and FSH levels return to pre-injection levels within this period. Histological data were tested for differences between groups with the Kruskal-Wallis one factor analysis of variance. RESULTS HORMONE PROFILES
Maturational changes (age; p < 0.01) in mean LH concentrations were evident ([able I). In addition, GnRH-A treatment influenced LH levels (treatment; p < 0.05) and the maturational profile (age x treatment; p < 0.01). In the control group plasma LH levels were high in the first 3 wk and declined thereafter until 9 wk. Increased levels were noted in the 10-14 wk period and values declined thereafter. Early treatment with GnRH-A reduced LH levels for 2 wk following drug administration. Late treatment with GnRH-A was without effect. Although all groups had elevated LH concentrations at 10-16 wk, levels were particularly high in the control group at 104
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