Active and passive immunization against luteinizing hormone in pigs

Active immunization of four adult pigs with highly purified porcine luteinizing hormone (pLH)--using method of multiside intradermal injections--has been performed and resulted in the production of specific antibodies. Immunization caused prolongation of estrous cycle to 47-49 days in two gilts and to 26 days in the other ones. Obtained anti-pLH pig serum was administered intravenously to 40 day pregnant gilt during 5 days (10 ml of serum, twice daily). Blood plasma progesterone (P4) concentrations decreased significantly from 8-13 to 2-4 ng/ml after two days of infusion and remained at this level for the next 5 days. Administration of this anti-pLH pig serum to gilt in the luteal phase of the estrous cycle caused the inhibition P4 to undetectable amounts. The different results were found after the passive immunization of 40 day pregnant gilt with rabbit anti-pLH globulin preparation (5 days, equivalent to 3 ml of original undiluted serum, twice daily). Although after two days of infusion P4 concentration decreased, in the next days P4 level slowly increased to pretreatment concentrations. The data suggest the possibility of specific anti-pLH antibody production in pigs by using active immunization, and the repeated utilization of such obtained antiserum in the same species for the inhibition of corpus luteum (CL) function.     

Active immunization against gonadotrophin-releasing hormone in Chinese male pigs: effects of dose on antibody titer, hormone levels and sexual development

The objective of this study was to determine the optimal dose of a GnRH vaccine for immunocastration of Chinese male pigs, based on immune, endocrine and testicular responses. Forty-two crossbred (Chinese Yanan x Large White) male pigs were randomly assigned to one of the five treatments as follows: (I) 0 microg (control, n=8); (II) 10 microg (n=8); (III) 62.5 microg (n=8); (IV) 125 microg (n=8); (V) 250 microg (n=10), D-Lys6-GnRH tandem dimer (TDK) peptide equivalent of conjugate (TDK-OVA), using Specol as the adjuvant. Pigs were immunized at 13 and 21 weeks of age and were slaughtered at 31 weeks of age. Blood samples for antibody titer and hormone assays were collected at 13, 21, 24 and 31 weeks of age. At these time-points, testis size was also measured. At slaughter, testis weight was recorded and fat samples were collected for androstenone assay. Four animals, one out of each immunized group, responded poorly to the immunization (non-responders). At slaughter, serum testosterone and LH levels, fat androstenone levels and testis size/weight of these non-responders were similar to those in control animals. Antibody titers of non-responders were substantially lower (P<0.05) than in other immunized pigs. For the animals that responded well to the immunization (immunocastrated pigs), serum testosterone and LH levels, fat androstenone levels and testis size or weight were reduced (P<0.05) as compared to either controls or non-responders, at all doses tested. There was a significant effect of dose of TDK-OVA on antibody titers. The overall mean antibody titers in the 62.5 or 125 microg dose group (53.6 and 50.5% binding, respectively) were significantly higher than in the 10 or 250 microg group (39.2 and 40.24% binding, respectively). At slaughter, there was a significant dose effect on testis size or weight and on serum testosterone levels, but there was no dose effect on serum LH levels and fat androstenone levels. Testis size or weight in the 10 microg group was reduced to a lesser extent (P<0.05) than in the three higher dose groups. At slaughter, in comparison to controls, mean testis size of immunocastrated pigs in treatments II-V was reduced to 55, 21, 33 and 25%, respectively, whereas testis weight was reduced to 39, 12, 18 and 14%, respectively. Reduction of testis size and/or weight is important for visual assessment of castration at the slaughterline, therefore, it is concluded that a dose of 10 microg peptide is not suitable. We conclude that, within the dose-range studied, the 62.5 microg dose is optimal for future GnRH immunization studies or future practical use in immunocastration of Chinese male pigs.     

Effects of active immunization against gonadotropin releasing hormone on serum luteinizing hormone,progesterone levels and estrous cycles in the guinea pig

Mature female guinea pigs that had been observed to undergo three consecutive periods of estrus at approximately 16-day intervals were immunized with either 100 μg gonadotropin releasing hormone (GnRH) conjugated to 100 μg bovine serum albumin (BSA) or 100 μg BSA alone during diestrus (day 5–10) of the fourth cycle. Booster immunizations were administered 32 days after the first injection. Animals were bled by cardiac puncture at the time of first injection and at 16, 32, 48 and 64 days. Animals were necropsied at 64 days after first treatment.Daily observation indicated that vaginal manifestation of estrus was not apparent after a period equal to one estrous cycle in seven of ten GnRH immunized guinea pigs and after two cycles in the remaining three GnRH immunized guinea pigs. Estrous cycles persisted in BSA treated females throughout the experiment.Serum luteinizing hormone (LH) declined significantly by 32 days after the first immunization against GnRH and remained lower than both pretreatment values and levels in control animals at the same bleeding times throughout the experiment. Serum progesterone levels were significantly lower in the GnRH immunized group than in the control group at 48 and 64 days.At necropsy the weight of the ovaries of GnRH immunized guinea pigs was significantly lower than that of controls. Corpora lutea and antral follicles were present in both GnRH treated and control females. The presence of serum progesterone levels and of antral follicles in the GnRH immunized females suggests that a low level of gonadotropic support may have persisted to 64 days after initiation of treatment.Results indicate that immunization against GnRH can reduce LH and progesterone levels and induce cessation of estrous cycles in the guinea pig.     

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of passive immunization against progesterone on its distribution in circulation and tissues of mice

Anti-progesterone monoclonal antibody, injected into mice 32 h after mating at a dose that blocks the establishment of pregnancy, produced a significant reduction in the concentration of progesterone in the ovary and uterus within 6 h after treatment. Uterine concentrations remained lower in treated compared with control animals for at least 24 h after injection. There was an associated transient increase in plasma LH and FSH concentrations, but there was no change in plasma prolactin values. The percentage of total progesterone in the circulation that was unbound was reduced after treatment, but the concentration of unbound progesterone was increased. Studies of antibody binding of steroid in the presence of uterine progesterone receptor protein showed that there was a stoichiometric relationship in the distribution of ligand between the two binders. The present findings suggest that the effects of passive immunization against progesterone are associated with perturbation of tissue concentrations of steroid in the target organ as a result of high antibody concentrations in the circulation.     

Serum cortisol levels affect the metabolic clearance rate of progesterone in female baboons.

Since interactions between progesterone (P₄), Cortisol (F), cortisone (E) and corticosteroid binding globulin (CBG) may influence the metabolic clearance rates (MCR) of these steroids, the effect of altering circulating F concentrations on clearance of the steroids was determined. MCR of P₄, F and E were determined by the iv constant infusion method in 6 pregnant and 6 nonpregnant baboons (Papio papio). Serum F concentrations were altered by iv infusion of 5 mg F/90 min or im injection of betamethasone (3 mg bi-daily for 2 days). Mean MCR-P₄ (1/d/kg ± SE) was greater (P < 0.01) in pregnant (92.8 ± 8.5) than in nonpregnant (53.9 ± 4.4) animals while mean MCR-F was similar in both groups ($\text{10.8 ± 1.2}\text{vs}\text{13.0 ± 1.5}$, respectively). Mean MCR-E was also similar in pregnant (30.8 ± 4.9) and nonpregnant (34.1 ± 4.5) baboons. Mean serum F concentrations (/gmg/100 ml ± SE) in 4 nonpregnant (42.0 ± 8.6) and 4 pregnant (52.2 ± 10.0) baboons were increased (P < 0.05) 60% by F administration but MCR-P₄, -F and -E were unaltered. Betamethasone treatment reduced (P < 0.05) serum F 75% in both groups. In nonpregnant baboons, betamethasone treatment reduced (P < 0.01) MCR-P₄ (37.3 ± 3.9), MCR-F (7.4 ± 1.6) and MCR-E (18.5 ± 3.7). Betamethasone treatment of pregnant animals reduced (P < 0.01) MCR-P₄ (56.5 ± 7.4), MCR-F (6.3 ± 0.8) and MCR-E (14.6 ± 2.6). Infusion of F into betamethasone-treated animals increased serum F levels and increased MCR-P4, -F and -E. It is concluded that variations in serum F levels affect the clearance of F, E and P₄ presumably because of the mutual interactions of these steroids with CBG.     

The effect of immunization against somatostatin on growth rates and growth hormone secretion in the chicken

The effect of both active passive immunization against somatostatin on growth rate and growth hormone levels was studied in chickens. Passive immunization against somatostatin by administration of antiserum had no effect on rate of growth of chickens and no persistent effect on circulating growth hormone (GH) levels. In acute experiments, administration of anti-somatostatin serum caused a marked elevation of GH levels in chickens at both 4 and 8 weeks of age, but the relative stimulation was greater in the older birds. Active immunization against somatostatin significantly stimulated growth rate in chickens, but was not shown to have a clear effect on circulating GH levels. These data suggest that somatostatin control over GH secretion may not be fully developed in the chicken at 4 weeks of age, but that immuno-neutralization of somatostatin can produce an increase rate of growth in chickens similar to that seen in mammals.     

Plasma estradiol 17-sulfate and 2-hydroxyestradiol 17-sulfate levels and their metabolic clearance rates in rats

By using highly specific antisera against estradiol 17-sulfate (E2-17-S) and against 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S), plasma concentrations of these sulfates in Wistar rats were determined. The plasma levels of E2-17-S and 2-OH-E2-17-S in the male were 23.5 +/- 5.3 and 21.6 +/- 6.2 pg/ml, respectively. During the estrus cycle of the female, the plasma concentration of E2-17-S reached its highest level 69.0 +/- 11.8 pg/ml, during the diestrus stage, and its lowest level 36.9 +/- 6.6 pg/ml, during the proestrus stage. Similar tendencies were observed in the case of 2-OH-E2-17-S. To examine the dynamic behavior of both sulfates, the plasma metabolic clearance rate (MCRp) of E2-17-S and 2-OH-E2-17-S were determined by infusion experiments. MCRp of E2-17-S and 2-OH-E2-17-S in male rats were 102 and 653 ml/h (means), respectively, and in female rats were 115 and 644 ml/h (means), respectively. The low MCRp values of both sulfates imply their slow metabolic turn-over.     

Active immunization of boars against gonadotrop in releasing hormone. I. Effects on reproductive parameters

Sexually mature boars were actively immunized against gonadotropin releasing hormone (GnRH) to characterize endocrine and gametogenic changes associated with immunoneutralization of endogenous GnRH. Injections of GnRH conjugated to bovine serum albumin (BSA) given five times over 24 wk induced production of antibodies against GnRH in all animals (n = 5). Active immunization against GnRH reduced serum concentrations of testosterone (P < 0.05) and luteinizing hormone (LH) (P < 0.05), testes volume (P < 0.01), paired testis weights (P < 0.05), paired epididymis weights (P < 0.05), sperm per testis (P < 0.01) and seminiferous tubule diameters (P < 0.001) when compared with controls (n = 4). These results indicate that both steroidogenic and spermatogenic functions are impaired in testes of mature boars actively immunized against GnRH.     

The secretion and metabolic clearance rates of growth hormone,insulin and prolactin in high- and low-yielding cattle at four stages of lactation

The basal plasma concentration, secretion rate (SR), distribution space (DS) and metabolic clearance rate (MCR) of GH, insulin and prolactin were measured in groups of 4 high- (HY) and 4 low-yielding (LY) cows at around 30, 90 and 150 days of lactation and after the cattle had been dried off. The kinetic parameters were determined using the single injection technique and measuring the disappearance of each hormone from the plasma by radioimmunoassay. Throughout lactation the DS for GH was higher in the HY cows (P<0.01) falling significantly in both groups when they were dry (HY, P<0.001; LY, P<0.01). There was no difference between the lactating group in the MCR of GH, but in both there was a significant (P<0.001) drop in the dry period which was accompanied by a fall in the SR (P<0.05). No differences were found in the DS and MCR of insulin between the groups during lactation but the average SR of insulin in the LY group was more than twice that in the HY group (P<0.05). Overall differences between stages of lactation were found in both groups for the SR (P<0.05), DS (P<0.01) and MCR (P<0.001) of insulin. None of the kinetic parameters differed for prolactin in the two groups. The DS (P<0.05) and MCR (P<0.001) of prolactin increased from day 30 to day 150 of lactation in both groups and then fell significantly (DS, P<0.05; MCR, P<0.01) when the animals were dry. In both groups the SR (P<0.01) and basal plasma concentration (P<0.01) of prolactin were higher in the dry period than at 30 and 90 days of lactation; probably due to the influence of season on the hormone.     

The Sulawesi Crested Black Macaque (Macaca nigra) menstrual cycle: changes in perineal tumescence and serum estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone levels.

Events in the normal menstrual cycle of the endangered Sulawesi Crested Black Macaque (Macaca nigra) were characterized. Daily blood samples were obtained during 10 menstrual cycles from five M. nigra demonstrating regular cycles. The amount of perineal tumescence was scored daily. Serum levels of estradiol and progesterone were determined by RIA, serum LH levels were determined by the mouse Leydig cell bioassay, and serum FSH levels were determined by the rat granulosa cell aromatase bioassay. Cycle length was 39.8 +/- 1.0 days (mean +/- SEM) with an LH surge occurring 25 +/- 1.5 days from the onset of menses. After menses, both LH and estradiol were initially depressed, with estradiol first exceeding 50 pg/ml 8 days before the LH surge. In five cycles, peak estradiol levels (340 +/- 44 pg/ml) occurred on the day of the LH surge (637 +/- 58 ng/ml) and in the other five cycles, on the day before the LH surge. There was a broad increase of FSH in midcycle without a well-defined surge corresponding to the LH surge. Progesterone began increasing on the day of the LH surge and reached peak levels (6.8 +/- 0.96 ng/ml) 8 days later. Maximal perineal tumescence was generally associated with the time of the LH surge, but variation between animals made it impossible to predict accurately the day of the LH surge by perineal tumescence scores alone.     

Changes in plasma progesterone, estradiol, follicle-stimulating hormone and luteinizing hormone during diestrus and ovulation in rats with 5-day estrous cycles: effect of antibody against progesterone

Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5-day cycle advances ovulation and completion of the cycle by 1 day in the majority of animals (75 and 80%, respectively). Progesterone (250 micrograms) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release.     

Metabolic clearance and production rates of oestradiol and progesterone during pubertal and postpubertal development in gilts

The crossbred gilts studied were aged 80 days (prepubertal), 180 days (prepubertal or postpubertal) and 260 days (postpubertal or pregnant). Estimates of metabolic clearance rate (MCR) of oestradiol and progesterone were consistently less (21 and 27%) in plasma than in blood, and these differences were not influenced by age of gilt. The MCR (1/day per kg body weight) for oestradiol and progesterone in plasma was greater (P less than 0.05) for 80-day-old prepubertal gilts than for older gilts. The MCR values of oestradiol and progesterone were similar in 180-day-old and 260-day-old gilts independent of reproductive state. Production rate (PR) of oestradiol and progesterone increased with age (80-180 days), and age and reproductive state differences were much more pronounced for PR of progesterone than of oestradiol. These results support the hypothesis that a reduction in the MCR and an increase in PR of oestradiol and progesterone in the gilt are associated with the process of pubertal development, and changes in gonadal steroid concentrations appear not to alter the MCR of oestradiol and progesterone.