Ovarian reaction and estrus manifestation in delayed puberty gilts after treatment with equine chorionic gonadotropin

Background

Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far.

Methods

Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy.

Results

The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface.

Conclusions

Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.     

Ovarian activity and uterus organometry in delayed puberty gilts

About 30% of the total number of gilts selected for reproduction at the large breeding farm units in Vojvodina (Republic of Serbia) are culled due to prolonged pre-insemination anoestrus (estrus not detected until 8 mo of age). The aim of this study was to provide the answer to the following question: do the culling gilts reach cyclic ovarian activity at all? One hundred seventy five culled gilts in which external estrus manifestations were not detected by 8 mo of age were sacrificed and their reproductive organs were examined for determination of sexual maturity (ovaries exhibiting pre-ovulatory follicles 8 to 11 mm in diameter, corpora hemorrhagica, corpora lutea and corpora albicantia). Uterine weights and horn length were also determined. Functional ovaries were observed in 107 (61.1%) examined gilts, with 62 animals having one and 45 having two puberty ovarian cycles (57.9% and 42.1%, respectively). Pathomorphological changes which could result in prolonged pre-insemination anoestrus were not observed on the reproductive organs of sexually mature gilts. Our results indicate that most of the culling gilts have reached cyclic ovarian activity. The main reason for culling due to the absence of external estrus manifestations in sexually mature gilts could be inadequate estrus detection technology.     

Ovarian response to human chorionic gonadotropin or gonadotropin releasing hormone in cats in natural or induced estrus

Human chorionic gonadotropin (hOG) or gonadotropin releasing hormone (GnRH) was given alone or with repeated coital stimuli to study ovarian activity and ovulation in the domestic cat. Adult cats in natural estrus (NE) or treated with follicle stimulating hormone (FSH-P) to induce estrus (2.0 mg/d for 5 d; IE) were assigned to one of five treatments: I, mating (M) only (three times daily for the first 3 d of estrus); II, M + hOG (250 IU, i.m. on Days 2 and 3 of estrus); III M + GnRH (25 mug, i.m. on Days 2 and 3 of estrus); IV, hOG only (250 IU, i.m. on Days 2 and 3 of estrus); or V, GnRH only (25 mug on Day 2 and 3 of estrus). Overall, IE females produced a greater (P < 0.05) number of corpora lutea (7.6 +/- 0.9) and unovulated follicles (18.9 +/- 2.1) than NE cats (4.9 +/- 0.6 and 3.6 +/- 0.9, respectively). For both NE and IE females, the M + hOG treatment (II) produced a greater number (P < 0.05) of ovulations (9.1 and 13.9, respectively) than any other ovulatory regimen (I, 4.1, 6.6; III, 4.1, 7.8; IV, 4.0, 6.2; V, 4.1, 5.6, respectively). These results indicate that 1) the excessive follicle number resulting from FSH-P treatment cannot be reduced with any of the hOG or GnRH treatments tested and 2) the use of hOG with copulatory stimuli synergistically enhances the ovulatory response of cats experiencing a natural estrus or those treated with FSH-P.     

Ovarian responses and embryo survival in recipient lactating Holstein cows treated with equine chorionic gonadotropin

The objectives of Experiment 1 were to determine a dose of eCG that would increase total luteal volume and plasma progesterone (P4) concentration on estrous cycle Day 7 in cows. The objectives of Experiment 2 were to determine the effects of treating embryo recipient lactating Holstein cows with eCG on pregnancy per embryo transfer (P/ET). In Experiment 1, lactating dairy cows at 63 ± 3 d postpartum (DIM) received no treatment (control, n = 10), or 600 (eCG6, n = 19), or 800 (eCG8, n = 19) IU of eCG 2 d after the start of the ovulation-synchronization protocol, Day -8 (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH). Blood was sampled on Days -10, -8, -3, 0, 7, and 14 for P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, 0, and 7. In Experiment 2, lactating dairy cows were paired according to parity and previous insemination (0 or > 1 insemination) and assigned to receive 800 IU of eCG (eCG8, n = 152) 2 d after the start of the ovulation-synchronization protocol (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH) or to receive no treatment (control, n = 162). Blood was sampled on Days -10, -3, 0, 7, and 14 for determination of P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, and 7, and cows with a CL > 20 mm in diameter on Day 7 received an embryo. In Experiment 1, P4 concentration on Day 7 was higher (P < 0.05) for eCG8 cows (2.3 ± 0.3 ng/mL) compared with control (1.2 ± 0.3 ng/mL) and eCG6 (1.1 ± 0.3 ng/mL) cows. In Experiment 2, eCG8 primiparous cows had more (P < 0.01) follicles > 10 mm on Day -3 compared with control primiparous cows (2.5 ± 0.9 vs 1.7 ± 0.5 mm), but multiparous control and eCG8 cows did not differ. A larger (P = 0.03) percentage of control cows received an embryo (87.5 vs 79.1%) compared with eCG8 cows. Among cows that received an embryo, total luteal volume on Day 7 was affected (P = 0.05) by treatment (eCG8 = 8.3 ± 0.4 cm³, control = 6.2 ± 0.4 cm³), but P4 concentration on Day 7 did not differ significantly between treatments. The percentage of cows pregnant 53 d after ET (overall, 24.2%) was not significantly different between control and eCG8 cows. In the current study, no differences in P/ET were observed between control and eCG8 cows and treatment with eCG increased the percentage of cows with asynchronous estrous cycle.     

The effect of luteinizing hormone, human chorionic gonadotropin and adrenocorticotropic hormone on puberty in gilts reared in confinement

Three trials were conducted to determine the effect of human chorionic gonadotropin (HCG), luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) on the incidence of estrus in gilts which were reared in confinement, relocated and exposed to a boar. In trial 1, 33 gilts were given saline or 250 IU HCG at an average age of 191 days and then relocated and observed for estrus twice daily for 10 days. Treatment with HCG did not increase the proportion of gilts that exhibited estrus. In trial 2, 42 gilts were relocated at an average age of 200 days. The gilts were assigned to three treatment groups and injected with saline, 68 mug LH or 1 mg LH. After 10 days of estrous detection, a laparoscopic examination of the ovaries was conducted on all gilts failing to exhibit estrus. In groups 1 to 3, the proportions of gilts exhibiting estrus or ovulating during the 10 days after treatment were 13 of 21, 6 of 10, and 5 of 11, respectively. In trial 3, 12 gilts were relocated to pasture lots, given saline or 80 IU ACTH twice daily for 2 days and checked for estrus for 14 days. The proportions of gilts that exhibited estrus after the administration of saline or ACTH were 4 of 6 and 6 of 6, respectively. The results indicate that the incidence of estrus in gilts reared in confinement, relocated and exposed to a boar was not affected by pre-treatment with exogenous HCG, LH or ACTH.     

Variations in the properties of equine chorionic gonadotropin

The objectives of this paper are to review the chemical and biological properties of equine chorionic gonadotropin (eCG, PMSG) isolated from the serum. Comparisons are made with eCG isolated from endometrial cups, trophoblast cell culture medium, and low titer serum. The results show that eCG can vary, depending on the source, in both chemical and biological (LH and FSH activity) properties.     

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.     

Effects of cloprostenol, human chorionic gonadotropin and estradiol benzoate treatment on estrus synchronization in dairy cows

Two consecutive experiments were conducted. In Experiment 1, 24 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received intramuscularly (i.m.) 500 mcg of cloprostenol, 1250 IU of human chorionic gonadotropin (hCG) and 5 mg of estradiol benzoate 12 h after cloprostenol treatment. Cows in Group II received 750 IU i.m. of hCG and 3 mg of estradiol benzoate 12 h after cloprostenol treatment. Treatment was given on Day 16 after estrus in both groups. All animals showed estrus within 24 to 48 h after cloprostenol treatment. The average interval from cloprostenol injection to the onset of estrus was not influenced by treatments. Four cows in Group I failed to ovulate and became cystic. In Experiment 2, 71 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received 500 mcg i.m. of cloprostenol after corpus luteum detection by palpation per rectum. Cows in Group II received 500 mcg of cloprostenol plus 750 IU of hCG and 3 mg of estradiol benzoate 12 h after. When estrus ready for service was confirmed by rectal examination, cows were inseminated. The percentage of cows ready for service tended to be lower (P < 0.06) between cows in Group I (88%) and those in Group II (100%). The average interval from cloprostenol treatment to service was longest (P < 0.001) in Group I (78.7 h +/- 14.9, X +/- SD) vs Group II (48 h +/- 2.9). The degree of readiness for service synchrony was lowest (P < 0.001) in Group I (59.3%) vs Group II (94.2%). The pregnancy rates of cows synchronized or treated were not altered by hCG-estradiol benzoate treatment (P > 0.25). These results suggest that in dairy cows treated with cloprostenol following palpation per rectum of a corpus luteum and then with 750 IU of hCG and 3 mg of estradiol benzoate 12 h later, a single fixed-time insemination at 48 h after cloprostenol treatment should be performed.     

The reaction of tetranitromethane with human chorionic gonadotropin.

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding.     

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.     

Ascertainment and treatment of delayed puberty

The majority of patients with pubertal delay, can be classified as having primary pubertal delay (constitutional delay of growth and puberty, CDGP), although any child with a chronic disease could present with delayed puberty. In contrast, children with hypogonadism, either hyper- or hypogonadotropic, exhibit a total absence of pubertal development. Hence, early evaluation of these patients should be performed. Delay of puberty leads to psychological problems, secondary to short stature and/or delay in the acquisition of secondary sex characteristics and the reduction of bone mass. Although the final height in patients with CDGP is usually normal, some of these patients do not reach the third percentile or remain in the lowest part of the growth chart according to familial height. The most common reason for treating CDGP patients, usually with sex steroids, is for psychological difficulties and for loss of bone mineralization. Treatment must be individualized. Therapeutic options and new drugs will be discussed. Appropriate treatment and adequate nutritional intake are indicated in patients with delayed puberty due to chronic illness. In patients with hypo- or hypergonadotropic hypogonadism, puberty must be induced or completed. Different treatments (GnRH analogues, gonadotropins and sex steroids), and the main objectives are discussed.     

The hormonal and clinical response of gilts with delayed puberty to GnRH: A preliminary study

Four gilts with delayed puberty were treated with 1 mg of Gn-RH intravenously. Three of the four gilts showed heat after treatment and heats recurred thereafter at normal intervals in these three pigs. All pigs responded with increased blood content of LH after injection of Gn-RH (1.45–6.1 ng/ml). The failure of one pig to exhibit heat after the injection of Gn-RH could not be explained by the resulting LH response.The results of this limited trial indicate that one cause of delayed puberty in gilts may be an insufficient gonadotropic release. Further studies are required to elucidate the reasons for the difference in sexual and ovulatory responsiveness of gilts with delayed puberty to Gn-RH.     

Ovarian response and embryo production in llamas treated with equine chorionic gonadotropin alone or with a progestin-releasing vaginal sponge at the time of follicular wave emergence

The objective of the study was to compare the ovulatory response and embryo production in llamas (Lama glama) treated with a single dose of equine chorionic gonadotropin (eCG) alone or combined with intravaginal medroxyprogesterone acetate (MPA) at the time of follicular wave emergence. Llamas with a growing follicle ≥7 mm in diameter were assigned to one of the following groups: (1) Control (n = 28): Nonstimulated llamas were mated and embryos were collected 7 d after mating. (2) eCG (n = 32): Llamas were given 5 mg luteinizing hormone (LH) (Day 0) to induce ovulation, 1000 IU eCG on Day 2, a luteolytic dose of prostaglandin F_2α on Day 6, mating on Day 7, and embryo collection on Day 14. (3) eCG+MPA (n = 34): Llamas were treated as those in the eCG group, but a sponge containing 60 mg MPA was placed intravaginally from Days 2 to 6. Llamas that did not respond to synchronization or superstimulation were excluded, leaving data from n = 26, 26, and 27 in the control, eCG, and eCG+MPA groups, respectively, for statistical analysis. The mean (±SD) number of follicles > 7 mm at the time of mating was greatest in the eCG group, intermediate in the eCG+MPA group, and lowest in the control group (16.6 ± 5.3, 12.9 ± 3.7, and 1.0 ± 0.0, respectively, P < 0.001). The number of corpora lutea was similar between eCG and eCG+MPA groups (10.1 ± 2.9 and 8.6 ± 3.7, respectively); both were higher (P < 0.001) than in controls (0.9 ± 0.3). The number of embryos did not differ significantly between the eCG and eCG+MPA groups (4.8 ± 2.8 and 3.5 ± 3.0, respectively), but both were higher (P < 0.001) than in the controls (0.7 ± 0.4). In conclusion, eCG, with or without MPA effectively induced a superovulatory response and multiple embryo production in llamas.     

Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).     

Induction of ovulation in gonadotropin treated gilts with synthetic gonadotropin releasing hormone

Four consecutive trials were conducted to investigate the possibility of controlling the time of ovulation in prepuberal gilts pretreated with PMS and HCG. In trial 1 it was shown that the GnRH analog Hoe 766 was superior to other compounds tested. The following trial revealed that 10 mug of that analog is the optimal dose to elicit an ovulatory response. In trial 3 it was found that the majority (73%) of gilts had started ovulating by 39 h after Hoe 766 injection. Individual gilts started ovulating up to 4 h sooner or up to more than 5 h later. Apparently the ovulatory process of an individual gilt extends over a period of 4 - 5 h. Double insemination of 9 gilts at 34 and 41 h after Hoe 766 resulted in fertilization rates and litter sizes that compared favourably with those of corresponding gilts treated with HCG.     

Response of dairy heifers to Prostaglandin F2alpha after treatment with human chorionic gonadotropin

After the observation of estrus following administration of Prostaglandin F(2alpha) (PGF(2alpha)), 79 dairy heifers were randomly either injected with 2500 IU of human chorionic gonadotropin (hCG) 48 h postestrus or maintained as controls with no injection at that time. Five to 9 d later, after a blood sample for progesterone determination was taken, all heifers were injected with 25 mg of PGF(2alpha). Heifers observed in estrus within the next 5 d were inseminated about 12 h after initial observation and were palpated for pregnancy 45 to 60 d postinsemination. Heifers treated with hCG had higher progesterone concentrations, reduced and delayed estrual responses, and lower insemination fertility rates when compared with control heifers.