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Infertility rates have risen significantly, one of which is due to monosodium glutamate (MSG) consumption. Recent studies have shown that flavonoids in black garlic (Allium sativum) act as antioxidants. The aim of this study was to assess the effect of black garlic extract (BGE) on gonadosomatic index, follicle-stimulating hormone (FSH) levels, and spermatozoa quality in rats exposed to MSG. Twenty-five healthy rats, aged ten to twelve weeks, were divided equally into five experimental groups: (1) negative control (NC), no intervention; (2) positive control (PC), fed with MSG 8 mg/kg; and (3) fed with MSG + BGE 200 mg/kg; (4) fed with MSG + BGE 400 mg/kg; and (5) fed with MSG + BGE 600 mg/kg. Oral MSG was administered once a day for two weeks before BGE administration was started for two weeks. The measured endpoints were gonadosomatic index, FSH levels, and spermatozoa concentration and quality (spermatozoa motility and abnormality). Analysis of variance (ANOVA) followed by Duncan's post hoc analysis was used to assess the measurement differences. The result suggested that the administration of BGE did not significantly affect the gonadosomatic index (p=0.513). Significant decreases in FSH levels (p=0.005) and spermatozoa concentration were observed in the PC group compared to other groups (p<0.001). Additionally, spermatozoa motility was significantly lower in the PC group compared to NC, BGE200, BGE400, and BGE600 (p<0.001), with higher motility noted in BGE200, BGE400, and BGE600 compared to PC (p<0.001). Furthermore, PC had significantly higher spermatozoa abnormalities compared to NC, BGE200, BGE400, and BGE600 (p<0.001). In conclusion, administration of BGE had a significant effect on the improvement of FSH levels and the quality of spermatozoa in rats exposed to MSG.

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Monosodium glutamate (MSG) is commonly used as a flavor-enhancing agent in foods, and studies have demonstrated its toxic effects in animal models. Black garlic is known for its antioxidant and anti-inflammatory properties; however, there is a lack of studies on the potential hepatoprotective effect of black garlic ethanol extract (BGE) against MSG-induced hepatotoxicity in rats. The aim of this study was to investigate the hepatoprotective effects of ethanol extract of black garlic against MSG-induced liver damage in animal model. Twenty-five male Wistar rats were randomly assigned to five groups (n=5): negative control, MSG only, and MSG with three different doses of BGE. The MSG only and MSG with BGE groups were orally administered with 8 mg/kg MSG daily. After MSG treatment, the MSG with BGE groups received BGE orally at daily doses of 200, 400, or 600 mg/kg body weight for 16 consecutive days. Subsequently, the levels of serum liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), interferon-gamma (IFN-γ), and cyclooxygenase-2 (COX-2) were measured. Our data indicated that the group treated with 200 mg/kg BGE had significant lower levels of AST and ALT significantly compared to the MSG-only group. The MSG-treated group had higher levels of the inflammatory markers COX-2 and IFN-γ, which were lowered by administration of 200 mg/kg BGE. In contrast, higher doses of BGE led to greater levels of COX-2 and IFN-γ compared to those in the MSG-only group. This study suggested that BGE might have hepatoprotective effects at low dose, potentially mitigating MSG-induced liver damage. However, the higher dose of black garlic extract did not alleviate inflammation, as shown by the higher levels of COX-2 and IFN-γ.

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This study was conducted to describe the stages of seminiferous epithelium (SE), determine the relative frequency of the stages, and identify the steps of spermatid development during spermatogenesis in the testicular tissue of Aceh bull. Seven pairs of the testicular organs of Aceh bull (Bos indicus) were used and then processed in a histological manner for staining using haematoxylin and eosin (H&E) and periodic acid-Schiff-haematoxylin (PAS-H). The stages of seminiferous tubules were examined using a tubular morphology method while spermatid development was observed based on the acrosome formation during spermatid development. Eight stages (stages I to VIII) of SE were found in the testicular seminiferous tubules of Aceh bull. Furthermore, the percentage of the relative frequency of each stage was 25.48, 15.38, 12.92, 4.74, 14.97, 10.69, 10.74, and 5.08%, respectively, with the relative frequency of premeiotic, meiotic, and postmeiotic phases being 53.78, 4.74, and 41.48%, respectively. Spermatid development from round to elongated spermatids occurred in 14 steps. Steps 1 to 7 were observed in stage I, steps 8 and 9 in stage II, steps 10 and 11 in stage III, step 12 in stage IV, step 13 in stages V and VI, and step 14 in stages VII and VIII. These findings can be used as a basis for further studies, particularly in evaluating the abnormality of the cellular composition of the seminiferous tubule in each stage of spermatogenesis and also in determining daily sperm production in Aceh bull.

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Background and Aim: The increase in the levels of the cortisol hormone caused by the stress conditions generated by an ovary transplantation procedure can damage the uterus of the transplant recipient as well as the transplanted ovaries. This study aimed to analyze the histopathological changes that occur in the uterine horn of pseudopregnant local rabbits (recipients), as well as the ovarian follicular integrity of the donor Aceh cattle after transplantation. Materials and Methods: After 30 days of adaptation, all rabbits were divided into three treatment groups: R1 (the group of rabbits that underwent ovarian transplantation for 3 days, n = 5), R2 (the group of rabbits that underwent ovarian transplantation for 5 days, n = 5), and R3 (the group of rabbits that underwent ovarian transplantation for 7 days, n = 5). Pseudopregnancy induction was performed using the pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) methods. The rabbits were injected with 100 IU of PMSG intramuscularly, followed by an injection of 75 IU of hCG intravenously 3 days later. Ovarian transplantation was performed on day 8 (day 0 was the day of hCG injection). The concentration of cortisol hormone metabolites was measured from fecal samples using an enzyme-linked immunosorbent assay technique. The uterus and ovaries were collected for histopathological and follicular dynamics examination after the transplantation process was completed. Results: The mean cortisol levels (ng/g) recorded before versus after the transplant in the R1, R2, and R3 groups were 146.23 ± 17.60 versus 338.84 ± 302.79, 128.97 ± 81.56 versus 174.79 ± 101.70, and 124.88 ± 43.61 versus 321.91 ± 221.63 (p < 0.05), respectively. The examination of the histopathological appearance of the uterus revealed edema in the uterine lumen, hyperemia and hemorrhage in the endometrium, necrosis of the epithelium, and infiltration of inflammatory cells. Hemorrhage and hyperemia were severe and filled the endometrium in the R1 compared with the R2 and R3 animals. Ovarian follicle development occurred in all treatment groups, although some histopathological features were observed. The number of tertiary follicles in R1, R2, and R3 animals was 24.67 ± 7.37, 20.67 ± 7.57, and 9.67 ± 3.79 (p < 0.05), respectively. Conclusion: Based on the results of this study, it can be concluded that the transplantation of ovaries from Aceh cattle into pseudopregnant local rabbits triggered an increase in the levels of the cortisol hormone and uterine histological changes; however, follicles were still detected at various stages of development in the transplanted Aceh cattle ovaries. The results of this study are valuable for clinicians and researchers because they provide information regarding an alternative in vivo ovarian preservation technique using pseudopregnant rabbits.

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AIM: This study aimed at determining the profiles of progesterone and bovine interferon-τ (bIFN-τ) and the correlation between the two in repeat breeding (RB) Aceh cattle and non-RB Aceh cattle. MATERIALS AND METHODS: The study was performed on five RB and five non-RB Aceh cows. These cows were subjected to estrous synchronization using the prostaglandin F2 alpha hormone, which was followed by artificial insemination (AI). Serum samples were collected on days 5, 6, 7, 15, 16, and 17 after AI to measure the concentration of progesterone at the beginning and end of the luteal phase and from days 14 to 18 after AI to measure the concentration of bIFN-τ. The concentrations of progesterone and bIFN-τ were determined using enzyme-linked immunosorbent assay. Pregnancy examinations were performed by ultrasonography on days 25, 35, 45, and 55 after AI. Data for progesterone and bIFN-τ concentrations were analyzed using the Mann-Whitney and t-tests, and the correlation between progesterone and bIFN-τ was analyzed using the Spearman correlation test. RESULTS: The average concentration of progesterone in RB Aceh cows tended to be lower than non-RB Aceh cows, but it was not significantly different (p>0.05). Similar results also found in the concentration of bIFN-τ which RB Aceh cows tended to have lower bIFN-τ concentrations compared to non-RB Aceh cows, but it was also not significantly different (p>0.05). Moreover, the concentrations of progesterone and bIFN-τ in RB and non-RB Aceh cows did not show a significant correlation (p>0.05). These results of the ultrasonography showed that non-RB Aceh cows were pregnant from day 25 to day 55 after AI, whereas RB Aceh cows were not pregnant and had early embryonic death. CONCLUSION: The concentrations of progesterone and bIFN-τ in non-RB Aceh cows tended to be higher than those in RB Aceh cows, although, it was not significantly different. Non-RB Aceh cows were able to maintain pregnancy until day 55, whereas RB Aceh cows were diagnosed with early embryonic death before day 25 after AI.

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BACKGROUND AND AIM: Testis (T) and epididymis (E) are waste from the abattoir that is rarely used. In fact, both organs contain important chemicals needed for spermatogenesis (e.g., hormones, proteins, and other molecules). Therefore, administration of a combination of testis and epididymis (CTE) extracts may activate androgen receptors (AR) and protein kinase A (PKA) molecules that play a prominent role in spermatogenesis. We, therefore, aimed at investigating the influence of the CTE extracts on the concentration of AR and PKA in male chicken. MATERIALS AND METHODS: This study used a completely randomized design with four treatment groups (K0, K1, K2, and K3) and five replications per group. K0 is a control group that received 1 mL normal saline, whereas K1, K2, and K3 are the test groups that received 1, 2, and 3 mL of CET extracts, respectively. Twenty male chickens (strain: broiler Mb 89), 3 weeks of age, weighing 500-700 g were used. We administered the injections in a 13-day period and on the 14th day; we collected and processed blood samples as serum to measure the AR and PKA concentrations using commercial chicken AR and PKA enzyme-linked immunosorbent assay kits, respectively. We performed analyses by analysis of variance using SPSS 20.0. RESULTS: The AR concentrations in K1, K2, and K3 groups increased by 4.26%, 10.97%, and 28.04%, respectively, compared to the K0 (control group). However, this increase was not significantly different between the groups (p>0.05). Moreover, the PKA concentrations increased by 2.97%, 2.60%, and 4.08% in K1, K2, and K3 groups, respectively, compared to the control group. However, this increase was not significantly different between the groups as well (p>0.05). CONCLUSION: The CTE extracts tended to increase the AR and PKA concentrations even though it is not significant. Therefore, it needs further study when using the CTE extracts for spermatogenesis in male chicken.

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BAKCGROUND AND AIM: Testis and epididymis are male reproductive organs that play an important role in spermatogenesis. These two organs are rich in the content of hormones and other molecules needed in the process of spermatogenesis which affect the quality of the spermatozoa. The objective of this study was to examine the effect of the administration of epididymis and testicular extracts and their combination on testosterone, pituitary adenylate cyclase-activating polypeptide (PACAP), and protamine 1 (PRM1) concentrations in the serum of male chicken. MATERIALS AND METHODS: Twenty male chickens (broiler strain Cp707), aged 3 weeks and weighing 800-1000 g, were randomly divided into four different groups including a control group (T0) = injected with 1 ml normal saline and treatment groups: T1 = injected with 1 ml epididymis extract, T2 = injected with 1 ml testicular extract, and T3 = injected with a combination of 1 ml epididymis + 1 ml testicular extract. The experiment was conducted for 13 days and at the end of the study (day 14), the chickens were sacrificed to obtain the serum. Furthermore, the concentrations of testosterone, PACAP, and PRM1 were then measured by using an enzyme-linked immunosorbent assay technique. RESULTS: The concentrations of PACAP and PRM1 did not show a significant difference between treatment groups (T1, T2, and T3) and control group (T0) (p>0.05). However, the concentration of testosterone showed a significantly higher difference in a group injected with a combination of 1 ml epididymis and 1 ml testicular extracts (T3) compared to the control group (T0) (p<0.05). CONCLUSION: The administration of epididymis and testicular extracts and their combination did not affect the increase of PACAP and PRM1 concentration. However, a combination of these extracts significantly affects the increase of testosterone concentration in the serum of male chicken.

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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 -20 OC (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.

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Stages of the seminiferous epithelium of the testis of the wild Javan muntjac (Muntiacus muntjak muntjak) in hard antler period were characterized based on the tubular morphology method. The number and the relative frequencies of seminiferous epithelium stages and the morphometry of germinal cell nuclei were identified microscopically. We identified eight stages of seminiferous epithelium in testicular tissue of the Javan muntjac and found that the relative frequencies of stages I to VIII were 14.87, 15.12, 17.75, 6.87, 7.37, 12.37, 13, and 12.62%, respectively. The diameter of the nuclei of germinal cells varied in each stage of seminiferous epithelium. Diplotene-stage primary spermatocytes had prominent and large nuclei ~8.97 ± 1.0 µm in stages III and IV. Pachytene primary spermatocytes appeared in most stages, except stage IV, whereas leptotene- and diplotene-stage primary spermatocytes were found in stages I and II, and III and IV, respectively. Round spermatids were observed in stages IV to VIII and in stage I but were absent in stages II and III, while elongated spermatids were observed in all stages except stage I. Our findings show that the stages of seminiferous epithelium in the Javan muntjac are similar to those found in neotropical cervids, small ruminants, and other domestic animals.

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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.

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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.

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