Characterization of Meloidogyne enterolobii intercepted from baobab (Adansonia digitata L.) seedlings from Thailand during Japanese import plant quarantine inspection.

In April 2019, baobab (Adansonia digitata L.) seedlings from Thailand, exhibiting galls on the roots, were intercepted during an import plant quarantine inspection at Chubu Centrair International Airport, Japan. Root-knot nematodes (RKNs) were extracted from the galled roots of baobab seedlings and identified by morphological, morphometrical and molecular methods as the guava root-knot nematode, Meloidogyne enterolobii Yang & Eisenback. The morphology and morphometrics of the intercepted population were similar to those of the original and subsequent descriptions of M. enterolobii. The sequences of D2-D3 of 28S rRNA, mtDNA intergenic COII-16S rRNA and COI genes obtained in this study matched well (99-100% similarity) with each of the gene sequences of M. enterolobii deposited in GenBank. Phylogenetic analysis of these genes revealed that the intercepted population clustered with M. enterolobii and clearly differed from other RKN species. To the best of our knowledge, this is the first report of M. enterolobii from baobab.

Enhanced light extraction efficiency of Eu-related emission from a nano-patterned GaN layer grown by MOCVD.

Eu-doped GaN is a promising material for the active layer in red light emitting diodes. Although the output power of LEDs based on GaN:Eu has been increasing by a combination of structural and growth optimizations, there is still a significant limitation resulting from a poor light extraction efficiency, typical for high refractive index materials. Here we studied nanostructuring of the top of the optical active layer by nano-cubes for enhancement of the light extraction efficiency, and its effect on the optical emission characteristics. By etching nano-cubes into the active layer, we observed an increase in directional light output power of Eu3+ ions of up to 60%, as well as a grating effect. Simultaneously, the absorption of excitation light into the optical active layer was improved, leading to a 12.8 times increase of output power per available Eu3+ ion.

Long-term feeding of hydroalcoholic extract powder of Lepidium meyenii (maca) enhances the steroidogenic ability of Leydig cells to alleviate its decline with ageing in male rats.

This study examined whether feeding hydroalcoholic extract of Lepidium meyenii (maca) to 8-week-old (sexually maturing) or 18-week-old (mature) male rats for more than a half year affects serum testosterone concentration and testosterone production by Leydig cells cultured with hCG, 22R-hydroxycholesterol or pregnenolone. Testosterone concentration was determined in the serum samples obtained before and 6, 12, 18 and 24 weeks after the feeding, and it was significantly increased only at the 6 weeks in the group fed with the maca extract to maturing rats when it was compared with controls. Testosterone production by Leydig cells significantly increased when cultured with hCG by feeding the maca extract to maturing rats for 27 weeks (35 weeks of age) and when cultured with 22R-hydroxycholesterol by feeding it to mature rats for 30 weeks (48 weeks of age). Overall testosterone production by cultured Leydig cells decreased to about a half from 35 to 48 weeks of age. These results suggest that feeding the maca extract for a long time to male rats may enhance the steroidogenic ability of Leydig cells to alleviate its decline with ageing, whereas it may cause only a transient increase in blood testosterone concentration in sexually maturing male rats.

Treatment with the MAPK kinase inhibitor U0126 during the first two hours of in vitro maturation improves bovine oocyte developmental competence.

This study examined the effects of treatment with U0126, which inhibits MAPK by inhibiting MAPK kinase, during the first 2 hr of in vitro maturation on bovine developmental competence and on gap junction (GAPJ) communication between the oocyte and cumulus cells. The percentage of oocytes developing to the blastocyst stage in the group treated with 5 µM U0126 (28%) was significantly higher than that in controls (15%, p < .05), while that in the group treated with 10 µM U0126 (18%) was not. Breakdown of the GAPJs was delayed in the group treated with 5 µM U0126 when compared to controls, as estimated by immunohistochemical examination of connexin 43, which is a primary constituent of the GAPJs. These results indicate that treatment with 5 µM U0126 during in vitro maturation delays GAPJ breakdown and improves bovine oocyte developmental competence.

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) enhances testicular gene expression of 3ß-hydroxysteroid dehydrogenase in rats.

Although feeding diets containing the extract powder of Lepidium meyenii (maca), a plant growing in Peru's Central Andes, increases serum testosterone concentration associated with enhanced ability of testosterone production by Leydig cells in male rats, changes in testicular steroidogenesis-related factors by the maca treatment are not known. This study examined the effects of maca on testicular gene expressions for luteinizing hormone receptor, steroidogenic acute regulatory protein and steroidogenic enzymes. Eight-week-old male rats were given the diets with or without (control) the maca extract powder (2%) for 6 weeks, and mRNA levels were determined by reverse transcription quantitative real-time PCR. The results showed that the testicular mRNA level of HSD3B1 (3ß-hydroxysteroid dehydrogenase; 3ß-HSD) increased by the treatment, whereas the levels of the other factors examined did not change. These results suggest that increased expression of 3ß-HSD gene may be involved in the enhanced steroidogenic ability by the maca treatment in rat testes.

Changes of plasma concentrations of insulin-like peptide 3 and testosterone, and their association with scrotal circumference during pubertal development in male goats.

Insulin-like peptide 3 (INSL3) has been used as a testis-specific biomarker for puberty in several species, but the secretory profile of INSL3 during pubertal development in small ruminants is unknown. Here we sought to determine the age-related changes in the plasma concentrations of INSL3 and testosterone and their association with scrotal circumference during pubertal development in five male Shiba goats. Blood samples and scrotal circumference measurement were taken every 2 weeks from week 10 to week 52 of each goat's lifespan. Based on the changes in scrotal circumference, data were grouped into early pubertal (10-22 weeks), late pubertal (22-34 weeks) and post-pubertal (34-52 weeks) categories. The plasma concentrations of testosterone and luteinizing hormone (LH) were measured by enzyme-immunoassays (EIAs), and we used a time-resolved fluorescence immunoassay (TRFIA) to measure plasma INSL3. The biweekly sampling showed that the plasma INSL3 secretions maintained a moderate increase during and after puberty, whereas the plasma testosterone secretions fluctuated over the same period. The comparison of the three age categories revealed a significant increase (p < 0.01) in the mean plasma INSL3 concentrations during the late and post-pubertal periods compared to the early pubertal period. There was no difference in the mean plasma testosterone concentrations between the early and late pubertal periods, but a significant increase (p < 0.01) was observed during the post-pubertal period compared to early and late pubertal periods. The mean plasma LH concentrations increased significantly (p < 0.05) from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods. A significant increase (p < 0.05) in the mean scrotal circumference from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods was observed. The R2 value of the best regression curves between scrotal circumference and INSL3 (0.513; p < 0.001) was higher than that between scrotal circumference and testosterone (0.162; p < 0.01) from 10 to 52 weeks of age. In conclusion, in male goats, plasma concentrations of INSL3 increased continuously during and after puberty, whereas testosterone secretions were fluctuated. The scrotal circumference was more highly correlated with the INSL3 concentrations than with testosterone, implying that INSL3 is superior as a biomarker of testicular total Leydig cell volume.

A novel LSD1 inhibitor NCD38 ameliorates MDS-related leukemia with complex karyotype by attenuating leukemia programs via activating super-enhancers.

Lysine-specific demethylase 1 (LSD1) regulates gene expression by affecting histone modifications and is a promising target for acute myeloid leukemia (AML) with specific genetic abnormalities. Novel LSD1 inhibitors, NCD25 and NCD38, inhibited growth of MLL-AF9 leukemia as well as erythroleukemia, megakaryoblastic leukemia and myelodysplastic syndromes (MDSs) overt leukemia cells in the concentration range that normal hematopoiesis was spared. NCD25 and NCD38 invoked the myeloid development programs, hindered the MDS and AML oncogenic programs, and commonly upregulated 62 genes in several leukemia cells. NCD38 elevated H3K27ac level on enhancers of these LSD1 signature genes and newly activated ~500 super-enhancers. Upregulated genes with super-enhancer activation in erythroleukemia cells were enriched in leukocyte differentiation. Eleven genes including GFI1 and ERG, but not CEBPA, were identified as the LSD1 signature with super-enhancer activation. Super-enhancers of these genes were activated prior to induction of the transcripts and myeloid differentiation. Depletion of GFI1 attenuated myeloid differentiation by NCD38. Finally, a single administration of NCD38 causes the in vivo eradication of primary MDS-related leukemia cells with a complex karyotype. Together, NCD38 derepresses super-enhancers of hematopoietic regulators that are silenced abnormally by LSD1, attenuates leukemogenic programs and consequently exerts anti-leukemic effect against MDS-related leukemia with adverse outcome.

Testicular gene expression of steroidogenesis-related factors in prepubertal, postpubertal, and aging dogs.

Developmental and aging changes in testicular factors related to steroidogenesis are unknown in dogs. Using reverse transcription quantitative real-time PCR, this study examined testicular mRNA levels of CYP11A1 (P450 cholesterol side-chain cleavage enzyme, P450scc), CYP17A1 (P450 17α-hydroxylase/C17-20 lyase, P450c17), HSD3B2 (3ß-hydroxysteroid dehydrogenase, 3ß-HSD), CYP19A (P450 aromatase, P450arom), STAR (steroidogenic acute regulatory protein, StAR), cyclooxygenase (COX) -1 and COX-2 in prepubertal (4-6 months of age), postpubertal (1 year of age), and aging (2-18 years of age) dogs. Testicular mRNA levels for P450scc, 3ß-HSD, StAR, COX-1, and COX-2 did not change from prepubertal to postpubertal stages, whereas that for P450arom markedly and abruptly increased and that for P450c17 gradually decreased. In postpubertal and aging dogs, a negative correlation was found between aging and testicular P450arom mRNA levels. Based on the rapid testicular growth observed during puberty, these results suggested that total testis gene expression for steroidogenesis-related factors, in particular for P450arom, increases during puberty in dogs. In addition, the decline in P450arom gene expression during aging may affect the ability to synthesize steroids in canine testes.

Effects of long-acting GnRH antagonist, degarelix acetate, on plasma insulin-like peptide 3, testosterone and luteinizing hormone concentrations, and scrotal circumference in male goats.

We recently reported that plasma insulin-like peptide 3 (INSL3) concentrations increased soon after endogenous and exogenous stimulations of LH in male goats and bulls. However, the effects of LH suppression on INSL3 secretion are unknown in domestic animals. Here, we examined the effects of a long-acting GnRH antagonist (degarelix acetate; 4 mg/kg) on the secretions of plasma INSL3 and testosterone in two phases, an immediate and a long-term phase in male goats (n = 6; aged, 13-16 months). During the immediate phase, blood was taken at 15-minute intervals for 8 hours on Days -5, 0, and 3. The GnRH antagonist was administered after 2-hour sampling of Day 0. Moreover, a daily blood sample was taken from Day 0 to Day 7, followed by twice a week until 9 weeks and finally at week 10. The scrotal circumference was recorded before treatment and continued biweekly until week 10. Concentrations of LH, INSL3, and testosterone in plasma were determined by EIA and the pulsatile nature of secretion analyzed using pulse XP software. The mean concentrations, pulse frequency (per hour), and pulse amplitude (peak-nadir) of plasma LH and testosterone reduced from pretreatment to posttreatment Day 0 and Day 3 (P < 0.05). A decline in mean concentrations, pulse frequency, and pulse amplitude of INSL3 was exhibited on posttreatment Day 3 compared with pretreatment (P < 0.01). During long-term sampling, a decline (P < 0.01) in plasma testosterone and INSL3 concentrations was observed 1 day after treatment and remained lower until 8.5 weeks after treatment, and thereafter returned to pretreatment levels. A reduction in scrotal circumference was recorded 4 weeks after treatment and remained lower until 10 weeks after treatment (P < 0.05). In conclusion, the acute regulation of INSL3 by LH was confirmed by reduction of plasma INSL3 levels within 3 days after GnRH antagonist treatment in male goats. Although the onset of suppression of testosterone was more rapid than that of INSL3, the low levels persisted for 8.5 weeks for both hormones, and subsequently the concentrations returned to pretreatment levels. A significant reduction in testicular size was also observed. The quick, long-lasting, and transient suppression of testosterone and INSL3 after a single injection implies a potential application of this antagonist in reversible long-term chemical castration in male goats.

Vonoprazan prevents bleeding from endoscopic submucosal dissection-induced gastric ulcers.

BACKGROUND: Vonoprazan, a potassium-competitive acid blocker, is expected to improve the healing of endoscopic submucosal dissection (ESD)-induced gastric ulcers compared with proton pump inhibitors (PPIs). AIM: To compare the healing status of ESD-induced gastric ulcers and the incidence of post-ESD bleeding between subjects treated with vonoprazan for 5 weeks and those treated with PPIs for 8 weeks. METHODS: Patients in the vonoprazan group (n = 75) were prospectively enrolled, whereas patients in the PPI group (n = 150) were selected for a 2:1 matched historical control cohort according to baseline characteristics including gastric ulcer size immediately following ESD, age, sex and status of Helicobacter pylori infection. Two controls per case of vonoprazan-treated group were matched with a margin of 20% in terms of ulcer size and a margin of 5 years in terms of their age. RESULTS: Although a higher number of completely healed ulcers was observed in the PPI group (95/150, 63.3%) than that in the vonoprazan group (14/75, 18.7%; P < 0.001), the ulcer size reduction rates, which were 96.0 ± 6.7% in the vonoprazan group and 94.7 ± 11.6% in the PPI group, were not significantly different (P = 0.373). The post-ESD bleeding incidence in the vonoprazan group (1/75, 1.3%) was less than that in the PPI group (15/150, 10.0%; P = 0.01). The factors affecting post-ESD bleeding incidence were the type of acid secretion inhibitor (P = 0.016) and use of an anti-thrombotic agent (P = 0.014). CONCLUSION: Vonoprazan significantly reduced post-endoscopic submucosal dissection bleeding compared with PPIs.

Fetal gender prediction based on maternal plasma testosterone and insulin-like peptide 3 concentrations at midgestation and late gestation in cattle.

We compared maternal plasma testosterone and insulin-like peptide 3 (INSL3) concentrations between dams carrying a male versus female fetus from early to late gestation and examined the application of maternal hormonal concentrations to fetal gender prediction in dairy and beef cattle. Blood samples were collected from Holstein cows or heifers (N = 31) and Japanese Black beef cows (N = 33) at 1-month intervals at 2 to 8 months of gestation. Fetal gender was confirmed by visual observation of external genitalia of calves just after birth. Plasma testosterone and INSL3 concentrations were determined by enzyme-immunoassay. Fetal genders were judged based on cutoff values of maternal testosterone and INSL3 concentrations (male, if it was ≥ cutoff value; female, if < cutoff value), which we set for each hormone at each gestational month using receiver operating characteristic curves. Plasma testosterone concentrations were higher for dams with a male fetus than those with a female at 4, 5, 7, and 8 months for the dairy cattle (P < 0.05) and at 4, 5, 6, and 8 months for the beef cows (P < 0.05). Plasma INSL3 concentrations were higher for dams with a male fetus than those with a female at 2 and 6 months for the dairy cattle (P < 0.05) and at 4 to 8 months for the beef cows (P < 0.05). The predictive values and detection rates for fetal gender prediction based on maternal testosterone concentrations were 75.8% to 79.3% for dairy cattle at 5 and 7 months and for beef cows at 5 and 6 months, whereas those values by maternal INSL3 concentrations were 71.0% to 72.4% for the dairy cattle at 6 months and beef cows at 4 and 8 months. When multiple time points of testosterone and INSL3 concentrations at several midgestation and late gestation months were considered for fetal gender prediction, predictive values were 89.3% (5-7 months) and 85.7% to 88.0% (4-6, 8 months) for the dairy and beef breeds, respectively. Maternal testosterone and INSL3 concentrations in dams carrying a male fetus were higher than those carrying a female at midgestation and/or late gestation in Holstein and Japanese Black beef cattle. Nearly, 80% accuracy was obtained for fetal gender prediction by a single time point of maternal plasma testosterone concentrations at midgestation. Nearly 90% accuracy for the prediction was obtained when multiple time points of testosterone and INSL3 concentrations from midgestation to late gestation were considered.

Acute regulation of plasma insulin-like peptide 3 concentrations by luteinizing hormone in male goats.

Recently, it was reported that in bulls secretion of insulin-like peptide 3 (INSL3) in blood occurred in a pulsatile manner and was acutely regulated by LH. In the present study, the acute regulation of plasma INSL3 and its temporal relationships with LH and testosterone were examined in six sexually matured male goats using the following experimental design. (1) After stimulating LH release by administering a GnRH analogue, blood levels of LH, INSL3, and testosterone were monitored at 15-minute intervals for 2 hours followed by hourly intervals up to 8 hours. (2) After activation of the LH receptor by hCG blood levels of INSL3 and testosterone were determine at 15-minute intervals for 2 hours, followed by hourly intervals up to 8 hours, daily intervals up to Day 8, and finally on Day 12. (3) The release of LH, INSL3, and testosterone in normal physiology was established at 15-minute intervals for an 8-hour session. Concentrations of LH, INSL3, and testosterone in plasma were measured by enzyme-immunoassays. After GnRH treatment, mean plasma concentrations of all three hormones increased (P < 0.05) dramatically from 30 minutes and remained high until 120 minutes (LH), 75 minutes (INSL3), and 4 hours (testosterone) after treatment. After hCG treatment, mean plasma INSL3 concentrations increased (P < 0.05) from 30 minutes and remained elevated until the end of sampling on Day 12. An increase (P < 0.05) in mean plasma testosterone concentrations occurred from 15 minutes and remained high until Day 6. The mean increase (maximum per pretreatment concentration) of INSL3 concentrations after administration of GnRH and hCG was lower (P < 0.01) than that of testosterone. The secretory pattern of LH, INSL3, and testosterone in the general circulation was pulsatile with a frequency of 5.5 ± 0.6, 4.7 ± 0.5, and 2.2 ± 0.5, respectively, during the 8-hour period. Twenty out of 28 (71%) of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse. The mean increase (peak per basal concentration) of INSL3 pulses (2.1 ± 0.1 fold, n = 28) was lower (P < 0.01) than that of testosterone pulses (4.3 ± 2.2 fold, n = 13). In conclusion, secretion of INSL3 in blood occurred, like in bulls, in a pulsatile manner soon after LH pulses in male goats. The absolute concentrations of INSL3 in male goats were higher than that reported in other mammals. Insulin-like peptide 3 concentrations were acutely increased by endogenous and exogenous LH in male goats, but the rise of INSL3 was lower than that of testosterone.

Production of feline leukemia inhibitory factor with biological activity in Escherichia coli.

Leukemia inhibitory factor (LIF) is a cytokine which is essential for oocyte and embryo development, embryonic stem cell, and induced pluripotent stem cell maintenance. Leukemia inhibitory factor improves the maturation of oocytes in the human and the mouse. However, feline LIF (fLIF) cloning and effects on oocytes during IVM have not been reported. Thus, we cloned complete cDNA of fLIF and examined its biological activity and effects on oocytes during IVM in the domestic cat. The aminoacid sequence of fLIF revealed a homology of 81% or 92% with that of mouse or human. The fLIF produced by pCold TF DNA in Escherichia coli was readily soluble and after purification showed bioactivity in maintaining the undifferentiated state of mouse embryonic stem cells and enhancing the proliferation of human erythrocyte leukemia cells. Furthermore, 10- and 100-ng/mL fLIF induced cumulus expansion with or without FSH and EGF (P < 0.05). The rate of metaphase II oocytes was also improved with 100-ng/mL fLIF (P < 0.05). We therefore confirmed the successful production for the first time of biologically active fLIF and revealed its effects on oocytes during IVM in the domestic cat. Feline LIF will further improve reproduction and stem cell research in the feline family.

Abnormal neutrophil scattergram obtained using Pentra MS CRP in the patients with myelodysplastic syndrome showing dysgranulopoiesis.

INTRODUCTION: Pentra MS CRP is an automated hematology analyzer capable of cytochemistry using Chlorazol black E, a lipid-staining agent, for white blood cell (WBC) differentials. Pentra MS CRP displays a WBC scattergram according to the cell volume obtained using flow impedance and light absorbance reflecting the Chlorazol black E (CBE)-positive lipid content. METHOD: Neutrophil scattergrams obtained using Pentra MS CRP were compared between 5 patients with myelodysplastic syndrome (MDS) and normal controls. Sudan black B (SBB)-staining patterns of peripheral blood neutrophils were subdivided into four types (types I, II, III, and VI) based on their staining intensity and scored by counting 200 cells. Such SBB scores were also compared between the two groups. RESULTS: Neutrophil scattergrams deviated downward in the MDS group, suggesting the decreased CBE positivity that seemed reflect the reduction of the lipid content in dysplastic neutrophils. SBB scores determined in this study were also decreased in the MDS group when compared with those in normal controls. CONCLUSION: Pentra MS CRP might rapidly generate useful information on dysplastic neutrophils in patients with MDS based on its cytochemistry for WBC differentials during routine laboratory hematology.

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) increases serum testosterone concentration and enhances steroidogenic ability of Leydig cells in male rats.

Although Lepidium meyenii (maca), a plant growing in Peru's central Andes, has been traditionally used for enhancing fertility and reproductive performance in domestic animals and human beings, effects of maca on reproductive organs are still unclear. This study examined whether feeding the hydroalcoholic extract powder of maca for 6 weeks affects weight of the reproductive organs, serum concentrations of testosterone and luteinising hormone (LH), number and cytoplasmic area of immunohistochemically stained Leydig cells, and steroidogenesis of cultured Leydig cells in 8-week-old male rats. Feeding the extract powder increased weight of seminal vesicles, serum testosterone level and cytoplasmic area of Leydig cells when compared with controls. Weight of prostate gland, serum LH concentration and number of Leydig cells were not affected by the maca treatment. The testosterone production by Leydig cells significantly increased when cultured with 22R-hydroxycholesterol or pregnenolone and tended to increase when cultured with hCG by feeding the extract powder. The results show that feeding the hydroalcoholic extract powder of maca for 6 weeks increases serum testosterone concentration associated with seminal vesicle stimulation in male rats, and this increase in testosterone level may be related to the enhanced ability of testosterone production by Leydig cells especially in the metabolic process following cholesterol.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls.

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at -0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and -0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.