Measuring hormone metabolites from feces is the most often used method to assess hormonal status in wildlife. Although immediate freezing of fecal samples collected in the field is the best method to minimize the risk of degradation of hormones over time, this is often not possible in remote field sites. Therefore, alternative storage and preservation methods for fecal samples are required in these conditions. We conducted an experiment to investigate if fecal glucocorticoid (FGCM) and progesterone metabolite (pregnanediol-3-glucuronide; PdG) levels measured from samples that were extracted with a simple, field-friendly methodology correlate with those generated from frozen samples. We also evaluated whether storing fecal samples in alcohol is a suitable alternative to preserve FGCM and PdG concentrations long-term (i.e. over a 9-month period) at locations where fecal extraction is not feasible. Finally, we tested if the hormone concentrations in unpreserved fecal samples of orangutans change over 14 h when stored at ambient conditions, representing the maximum duration between sample collection and return to the camp. FGCM and PdG levels measured from samples that were extracted with the field-friendly method showed strong correlations with those generated from frozen samples, and mean levels did not differ significantly between these methods. FGCM concentrations showed no significant change compared to control samples when fecal samples were stored for up to 6 months in alcohol at ambient temperature and PdG concentrations even remained stable for up to 9 months of storage. FGCM concentrations of fecal samples kept at ambient temperature for up to 14 h post-defecation did not significantly differ compared to control samples frozen immediately after collection. These results provide the basis for the successful monitoring of the physiological status of orangutans living in remote natural settings, like those included in the Indonesian reintroduction programs.

Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, we test the reliability of C and T concentration measurements in the serum of Aceh cattle, obtained using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, we evaluate the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C excretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups).   Results: Parallelism, accuracy, and precision tests showed that both  C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.05). Concentrations of T in bulls were significantly higher than in cows (p<0.05). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

This study aimed to investigate the relationship between cervical mucus viscosity and estrogen concentration, several blood biochemical profiles, and blood macro-mineral levels, as well as to determine the effect of cervical mucus viscosity and pH differences on pregnancy rates in Aceh cattle. Eight females Aceh cows aged 3-7 years old, with body weights of 150-250 kg were synchronized using a 25 mg single dose injection of PGF2α. Afterward, observation of estrus signs was performed for 30 minutes at 06:00 a.m. and at 06:00 p.m. Blood and cervical mucus samples were collected during the first cervical mucus secretion. The estrogen concentrations of each type of cervical mucus, namely thick, moderate, and thin were 29.39±6.29; 23.24±4.62; and 30.93 pg/mL, respectively. The pregnancy rate in cows with a cervical mucus pH of 6 and 9 was 0%. Meanwhile, the groups with a cervical mucus pH 7 and 8 had a pregnancy rate of 50%. An examination of cervical mucus showed different consistencies: five cows had mucus with a thick viscosity, with a 20% pregnancy rate; two had mucus with a moderate viscosity, with a 50% pregnancy rate and one had mucus with a thin viscosity, with a 100% pregnancy percentage. In conclusion, there is no significant relationship between estrogen concentration and cervical mucus viscosity levels in Aceh cattle. Levels of total protein, total cholesterol, and glucose were correlated to the viscosity of the cervical mucus. Higher levels of blood macrominerals (Na, K, Cl, Ca, P, and Mg) in female Aceh cattle resulted in greater viscosities of cervical mucus. The groups with cervical mucus pH of 7 and 8 had higher rates of pregnancy (50%) than the groups with cervical mucus pH of 6 and 9. The viscosity of cervical mucus also had an effect on the success of pregnancy in Aceh cattle.

The objective of this study was to examine the effects of repeated freeze-thaw cycles and short-term storage of fecal extracts at ambient temperature on the stability of fecal glucocorticoid (fGCM) and estrogen metabolite (fEM) levels from crested macaques.In total 100 aliquots of fecal extracts from fecal samples collected from female crested macaques (Macaca nigra) living at the Tangkoko-Batuangus Nature Reserve, North Sulawesi were used. We performed two different experiments: (1) An experiment to investigate if levels of fGCM and fEM measured from fecal extracts that were exposed to two, four, six and eight repeated freeze-thaw cycles (test groups) differ to control samples (i.e. fecal extracts always stored frozen); (2) An experiment to evaluate whether storing fecal extracts at ambient temperature for two, four, six, and eight days (test groups)affects the levels of fGCM and fEM compared to the control group (i.e. fecal extracts frozen immediately).Results showed that hormone levels were significantly increased (P<0.05) after four freeze-thaw cycles for fGCM and after eight freeze-thaw cycles for fEM. By contrast, there was no significant difference (P>0.05) in levels of fGCM and fEM between the test groups and the control group in fecal extracts stored at ambient temperature. In conclusion, our data show that more than two and six repeated freeze-thaw cycles should be avoided when measuring fGCM and fEM in crested macaque fecal extracts, respectively. We also demonstrate that storing fecal extracts at ambient temperature is possible for at least 8 days without taking a risk of affecting the stability of fGCM and fEM levels.

This study was conducted to examine testosterone concentrations its relationship with the scrotal circumference and physical characteristics of semen in aceh bulls. Semen samples were collected weekly from jugular vein of three aceh bulls aged 4-5 years old for 10 weeks. Testosterone concentration was measured by enzyme-linked immunosorbent assay (ELISA) method. Semens were collected by using artificial vagina and evaluated for physical characteristics namely ejaculatory volume, pH, and sperm motility, concentration, and abnormalities. Data were analyzed using correlation-regresion test. Testosterone concentrations showed a positive correlation with scrotal circumference (r = 0.799), number of sperm (r = 0.703), sperm motility (r = 0.857) and sperm abnormalities (r = -0,877). No correlation, however, was found between testosterone concentrations with semen volume (r = 0.038) and pH (r = 0.418). It can be concluded that testosterone concentrations correlated positively with scrotal circumference, numbers of sperm, sperm of motility and sperm of abnormality.

This study was aimed to determine the effect of epididymis extract (EE) on the testosterone and dihydrotestosterone (DHT) level of local male goat. An experimental study was performed using a completely randomized design (CRD) pattern of one-way analysis of variance (ANOVA). 15 local male goats aged 1.5 years with body weight 14-16 kg were used in this study. The K0 group as a control group, injected with only 1 ml physiological saline, while each KP1, KP2, KP3, and KP4 groups treated with multilevel EE dose, ie 1, 2, 3, and 4 ml / goat for 13 consecutive days. At the end of treatment (day 14th), testes, epididymis (caput, corpus, and cauda) and ductus deferens samples were taken through the close-castration method for examining the testosterone and DHT concentration by using enzyme-linked immunosorbent assay (ELISA) technique. Data gathered were later analyzed using ANOVA followed by Tukey’s HSD in SPSS 16.0 for Windows. The result showed that the average concentration of testosterone on K0, KP1, KP2, KP3, and KP 4 in testis respectively were 10.00±2.64 ng/ml; 7.66±2.51 ng/ml; 10.00±6.55 ng/ml; 0.66±0.57 ng/ml; 11.66±7.37 ng/ml; caput epididymis; 5.00±1.73 ng/ml; 2.33±1.52 ng/ml; 5.00±2.64 ng/ml; 1.33±0.57 ng/ml; 5.66±1.15 ng/ml; corpus epididymis; 1.33±0.57 ng/ml; 0.66±0.57 ng/ml; 4.00±2.64 ng/ml; 0.66±0.57 ng/ml; 4.33±2.30 ng/ml; cauda epididymis: 1.00±0.00 ng/ml; 0.66±0.57 ng/ml; 1.66±0.57 ng/ml; 1.00 ± 0.00 ng/ml; 2.00±1.73 ng/ml; ductus deferens: 3.66±2.51 ng/ml; 0.66±0.57 ng/ml; 3.00±1.00 ng/ml; 1.00±0.00 ng/ml and 3.66±1.15 ng/ml. While the average concentration of DHT on K0, KP1, KP2, KP3, and KP 4 in testis respectively; 10.00±2.64 ng/ml; 7.66±2.51 ng/ml; 10.00±6.55 ng/ml; 0.66±0.57 ng/ml; 11.66±7.37 ng/ml; caput epididymis; 5.00±1.73 ng/ml; 2.33±1.52 ng/ml; 5.00±2.64 ng/ml; 1.33±0.57 ng/ml; 5.66±1.15 ng/ml; corpus epididymis; 1.33±0.57 ng/ml; 0.66±0.57 ng/ml; 4.00±2.64 ng/ml; 0.66±0.57 ng/ml; 4.33±2.30 ng/ml; cauda epididymis: 1.00±0.00 ng/ml; 0.66±0.57 ng/ml; 1.66±0.57 ng/ml; 1.00 ± 0.00 ng/ml; 2.00±1.73 ng/ml; ductus deferens: 3.66±2.51 ng/ml; 0.66±0.57 ng/ml; 3.00±1.00 ng/ml; 1.00±0.00 ng/ml and 3.66±1.15 ng/ml. Statistical analysis showed that the administration of EE only increased testosterone concentration in testes had significant effect (P< 0.05). From this study, it can be concluded that the EE has the potential to improve spermatogenesis and sperm quality through increasing the testosterone concentration in the local male goats.

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C secretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups).   Results: Parallelism, accuracy, and precision tests showed that both  C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.01). Concentrations of T in bulls were significantly higher than in cows (p<0.01). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.