Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility.

Background and Aim: Fertility is crucial for enhancing the efficiency of livestock production, as it directly impacts the reproductive rates. A comprehensive understanding of the relationship between sperm quality and embryo development is key to optimizing reproductive outcomes and improving the quality of livestock. This study analyzed the developmental competence of in vitro embryos recovered from Bali cattle with normal or poor sperm motility. Materials and Methods: Nine bulls with normal fresh semen (NFS) or poor fresh semen (PFS) motility were ejaculated for semen. Semen ejaculates, including volume, motility, and sperm concentration, were evaluated immediately after collection to measure the quality of the fresh semen. Frozen semen was evaluated using computer-assisted semen analysis (CASA) for motility, progressive sperm motility, distance curve path, distance curve linear, distance straight line, average path velocity, curvilinear velocity, linear velocity, straightness (STR), linearity of forward progression (LIN), wobble, and average lateral head displacement (ALH). Bull groups were used to determine in vitro embryo cleavage ability after fertilization of Bali cattle. Ovaries of Bali cattle were collected by slicing, and only cytoplasmic oocytes with compact cumulus cells were used in this study. The oocytes were matured, and in vitro fertilization was performed using fertilization media with a final sperm concentration of 1.5 × 106 spermatozoa/mL. After 48 h, the embryo cleavage ability of the cultured oocytes was evaluated. Results: There were significant differences in motility values between the NFS and PFS groups; however, there were no significant differences in the volume or sperm concentration. There was a significant difference in the LIN value between the groups but no significant differences in other CASA parameters. There were no significant differences in the cleavage rate and morula between the groups, but a positive correlation was observed between the cleavage rate and the morula and between the morula and ALH. A significant negative correlation was observed between the cleavage rate and STR and between the morula and STR; no significant differences were observed for other variables. Conclusion: Despite variations in sperm characteristics, both normal and poor sperm motility demonstrated comparable in vitro embryonic development competence. These findings provide important insights into the fertility potential of Bali bulls, providing valuable information that can enhance selection strategies to improve the quality of livestock production.

Bovine nucleoprotein transitions genes and protein abundance as valuable markers of sperm motility and the correlation with fertility.

Bovine nucleoprotein transitions (TNPs), specifically TNP1 and TNP2, are essential molecules in sperm nucleus rich in arginine and lysine. These molecules act in the phase between histone expulsion and before incorporation of protamine in the spermatid nucleus. Therefore, this study aimed to analyze genes and protein abundance of TNP1 and TNP2 in sperm to determine the potential as motility markers and correlation with fertility in the field. An objective evaluation method, CASA-Sperm Vision, was used to separate 22 bulls into two groups (mg-A and mg-B) based on their increasing motility. Sperm quality parameters were also examined including velocity, mitochondrial membrane potential (MMP) by the JC-1 method, head defects using William staining, and DNA fragmentation by Halomax. TNPs genes abundance was performed using the RT-qPCR method, and the protein abundance was examined with the EIA approach. The fertility rate was also analyzed based on the conception rate generated from each bull in the field, with the data obtained from iSIKHNAS. The results showed that TNPs genes and protein abundance were significantly higher (P < 0.05) in mg-A compared to mg-B, followed by various sperm quality parameters and fertility rates (P < 0.05). Positive correlations were found in TNPs genes and protein abundance with motility, velocity, MMP, and fertility (P < 0.01). Meanwhile, a negative correlation (P < 0.01) was found between head defects and DNA fragmentation. These results showed the potential of TNPs as sperm motility markers and bull fertility.

Effects of Tourist and Researcher Presence on Fecal Glucocorticoid Metabolite Levels in Wild, Habituated Sulawesi Crested Macaques (Macaca nigra).

Ecotourism managers and researchers often assume that apparently habituated primate groups no longer experience adverse consequences of prolonged exposure to tourists or researchers. We examined the effects of tourists and researchers on fecal glucocorticoid metabolite output (FGCM) in three critically endangered, wild crested macaque (Macaca nigra) groups in Tangkoko Nature Reserve, Sulawesi, Indonesia. We assayed FGCM from 456 fecal samples collected from thirty-three adults. Tourists can walk through and among macaque groups freely. Hence, we examined the possible effects of tourists both (1) in the reserve when away and not interacting with the study groups and (2) when they were present within the macaque groups. Generalized Linear Mixed Model (GLMM) analysis indicated that when tourists were present in the forest, but not directly among the macaques, FGCM levels in the macaque tourism groups were higher in months with more tourists. When tourists were among the macaque groups, some female macaques experienced rises and subsequent postexposure decreases in FGCM levels, consistent with predictions for acute stress. Male FGCM levels increased with tourist numbers within the group. Nevertheless, they were not significantly different from levels during undisturbed or postexposure conditions. FGCM responses related to researchers in groups varied by group, sex, and tourist presence. However, the temporal patterning of FGCM responses showed little evidence of chronic stress from tourism at this site.

Sperm Head Morphology Alterations Associated with Chromatin Instability and Lack of Protamine Abundance in Frozen-Thawed Sperm of Indonesian Local Bulls.

This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus's stability and induce morphological alterations in the sperm head.

Correlation between Post-Thaw Spermatozoa Quality of the Endangered Javan Banteng with OPN Gene Expression.

The role of ex situ conservation facilities or captivity through captive breeding programs is essential in the conservation of the endangered Javan banteng. The development of semen cryopreservation may assist on one side of the conservation plan. However, the male Javan banteng reproductive capability must be considered as it influences the targeted outputs. Studying the potential biomarker for fertility such as osteopontin gene expression is also expected to help predict male fertility. Therefore, this study aimed to analyze the quality of spermatozoa after thawing to help predict the male reproductive capability of Javan banteng. Furthermore, this study investigated the potential role of osteopontin gene expression in male Javan banteng fertility. A positive reinforcement approach was used to accustom the male and female animals as we focused on establishing a collection procedure using neither sedation nor anaesthesia. Semen samples were collected at Taman Safari Indonesia, Bogor, in accordance with the female banteng receptivity. Semen samples were then evaluated and then cryopreserved under field conditions. Our study showed the different predicted reproductive capability of the Javan banteng based on the post-thaw spermatozoa quality, which showed significant differences. The OPN gene showed positive correlations with the progressive motility (r = 0.711, p = 0.048), viability (r = 0.822, p = 0.012), and acrosomal integrity (r = 0.665, p = 0.072) of Javan banteng spermatozoa after thawing. Our study demonstrated the predicted Javan banteng reproductive capability based on various post-thaw spermatozoa variables. This finding is also the first report on the OPN gene potential to be developed as the assessment tool of post-thaw spermatozoa quality of the male Javan banteng. The findings in our study may help give recommendations for future breeding programs, especially in the ex situ conservation sites.

Ten Years of Positive Impact of a Conservation Education Program on Children's Knowledge and Behaviour Toward Crested Macaques (Macaca nigra) in the Greater Tangkoko Area, North Sulawesi, Indonesia.

In areas where primates are threatened, environmental education interventions are a key way to increase the local population's knowledge of their environment and encourage positive attitudes and habits to preserve the environment and wildlife on a local and global scale. This study assesses the impact of Tangkoko Conservation Education (TCE), the Macaca Nigra Project's conservation education programme, running since 2011 in North Sulawesi for school children, teachers, and the local population. TCE's goal is to help people increase their knowledge of their environment and develop more positive habits and behaviours towards their local environment, especially the Critically Endangered and endemic crested macaques (Macaca nigra). We measured the efficacy of TCE's programme for pupils using questionnaires provided one month before and one month after a year-long conservation education programme at school. Pupils' knowledge and behaviour scores increased significantly after their participation in the programme. Their habits score also increased but this increase was not statistically significant. Girls scored significantly higher than boys in terms of positive behaviour towards wildlife. Children participating in the programme more than once seemed to obtain higher scores in their second participation, although the sample size was too small for formal analysis. Despite some limitations, this study demonstrates the positive impact of our programme on young people living in an area where primates are threatened. We hope that this research will inspire similar programmes in Sulawesi and elsewhere by providing methods and activities to help prevent primate extinction. Supplementary Information: The online version contains supplementary material available at 10.1007/s10764-023-00356-9.

Crested macaque facial movements are more intense and stereotyped in potentially risky social interactions.

Ambiguity in communicative signals may lead to misunderstandings and thus reduce the effectiveness of communication, especially in unpredictable interactions such as between closely matched rivals or those with a weak social bond. Therefore, signals used in these circumstances should be less ambiguous, more stereotyped and more intense. To test this prediction, we measured facial movements of crested macaques (Macaca nigra) during spontaneous social interaction, using the Facial Action Coding System for macaques (MaqFACS). We used linear mixed models to assess whether facial movement intensity and variability varied according to the interaction outcome, the individuals' dominance relationship and their social bond. Movements were least intense and most variable in affiliative contexts, and more intense in interactions between individuals who were closely matched in terms of dominance rating. We found no effect of social bond strength. Our findings provide evidence for a reduction in ambiguity of facial behaviour in risky social situations but do not demonstrate any mitigating effect of social relationship quality. The results indicate that the ability to modify communicative signals may play an important role in navigating complex primate social interactions. This article is part of the theme issue 'Cognition, communication and social bonds in primates'.

Temporal dynamics and fitness consequences of coalition formation in male primates.

Coalition formation is one of the most striking forms of cooperation found in animals. Yet, there is substantial variation between taxa regarding the mechanisms by which coalitions can result in fitness consequences. Here, we investigate the influence of coalitions on dominance rank trajectories and subsequently on reproductive success in wild male crested macaques (Macaca nigra) at Tangkoko Nature Reserve (Sulawesi, Indonesia). We observed 128 coalition events involving 28 males and tested how a variety of coalition properties and factors related to the social environment influenced future male rank. We further used genetic paternity analysis of 19 infants conceived during the study to assess male reproductive success. Our results show that males participating in coalitions achieved higher-than-expected future ranks, while coalition targets had lower-than-expected future ranks. Additionally, all-up coalitions had stronger effects on rank than all-down and bridging coalitions, and these were modulated by the relative strength of coalition partners versus targets. Finally, higher ranking males were more likely to sire infants than lower ranking males. These results provide important insights regarding the mechanisms underlying coalition formation and support the idea that one major path by which coalitions can affect fitness is through influencing male dominance trajectories.

PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls.

Functional genes and proteins in sperm play an essential role in bulls' reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

The potential of sperm bovine protamine as a protein marker of semen production and quality at the National Artificial Insemination Center of Indonesia.

BACKGROUND AND AIM: Protamine (PRM) is the major protein in the sperm nucleus and plays an essential role in its normal function. Moreover, PRM has great potential as a protein marker of semen production and quality. This study aimed to assess the potential of sperm bovine PRM as a protein marker of semen production and quality in bulls at the National Artificial Insemination (AI) Center of Indonesia. MATERIALS AND METHODS: The semen production capacity of each bull was collected from frozen semen production data at the Singosari AI Center for 6 months, and was then divided into two groups (high and low). A total of 440 frozen semen straws from six Limousin (LIM), six Friesian Holstein (FH), six Peranakan Ongole (PO), and four Aceh bulls aged 4-5 years were used in the study. The frozen semen was used to measure the concentration of PRM1, PRM2, and PRM3 using the enzyme immunoassay method. The frozen semen was also used to assess the quality of the semen, including progressive motility (PM) through computer-assisted semen analysis, sperm viability through eosin-nigrosin analysis, and the DNA fragmentation index through Acridine Orange staining. RESULTS: PRM1 was significantly higher in all bull breeds included in the study (p<0.00), followed by PRM2 (p<0.00) and PRM3 (p<0.00). PRM1 significantly affected semen production in LIM, FH, PO, and Aceh bulls (p<0.05). Moreover, PRM2 significantly affected semen production only in FH and Aceh bulls (p<0.05), whereas PRM3 affected this parameter in PO and Aceh bulls exclusively (p<0.05). Consistently and significantly, PRM1 was positively correlated with the PM and viability of sperm and negatively associated with its DNA fragmentation in LIM, FH, PO, and Aceh bulls (p<0.05; p<0.01). The correlation analysis between PRM2 and PRM3 and semen quality parameters varied across all bull breeds; some were positively and negatively correlated (p<0.05; p<0.01), and some were not correlated at all. CONCLUSION: PRM1 has excellent potential as a protein marker of semen production and quality in bulls at the National AI Center of Indonesia.

Female fertile phase synchrony, and male mating and reproductive skew, in the crested macaque.

High social status is the primary determinant of reproductive success among group-living male mammals. Primates living in multimale-multifemale groups show the greatest variation in the strength of this link, with marked variation in reproductive skew by male dominance among species, dependent on the degree of female fertile phase synchrony, and the number of competing males. Here, we present data on two groups of wild crested macaques (Macaca nigra), living in the Tangkoko Reserve, Sulawesi, Indonesia. We investigated male monopolization of fertile females in 31 cycles of 19 females, and genetic paternity of 14 offspring conceived during the study period. We show that female fertile phase synchrony was low, that females had few mating partners in their fertile phase, and that dominant males monopolized a high proportion of consortships and matings, resulting in marked and steep mating and reproductive skew. We conclude that female cycle asynchrony provides the opportunity for strong direct male-male competition in crested macaques, resulting in monopolization of females by dominant males, consistent with their marked sexual dimorphism. Our study provides a test of the underlying factors that determine the relative occurrence and strength of different mechanisms of sexual selection, and the phenotypes that evolve as a result.

The cost of associating with males for Bornean and Sumatran female orangutans: a hidden form of sexual conflict?

ABSTRACT: Sexual coercion, in the form of forced copulations, is relatively frequently observed in orangutans and generally attributed to their semi-solitary lifestyle. High ecological costs of association for females may be responsible for this lifestyle and may have prevented the evolution of morphological fertility indicators (e.g., sexual swellings), which would attract (male) associates. Therefore, sexual conflict may arise not only about mating per se but also about associations, because males may benefit from associations with females to monitor their reproductive state and attempt to monopolize their sexual activities. Here, we evaluate association patterns and costs for females when associating with both males and females of two different orangutan species at two study sites: Suaq, Sumatra (Pongo abelii), and Tuanan, Borneo (Pongo pygmaeus wurmbii). Female association frequency with both males and females was higher in the Sumatran population, living in more productive habitat. Accordingly, we found that the cost of association, in terms of reduced feeding to moving ratio and increased time being active, is higher in the less sociable Bornean population. Males generally initiated and maintained such costly associations with females, and prolonged associations with males led to increased female fecal cortisol metabolite (FCM) levels at Tuanan, the Bornean population. We conclude that male-maintained associations are an expression of sexual conflict in orangutans, at least at Tuanan. For females, this cost of association may be responsible for the lack of sexual signaling, while needing to confuse paternity. SIGNIFICANCE STATEMENT: Socioecological theory predicts a trade-off between the benefits of sociality and the ecological costs of increased feeding competition. Orangutans' semi-solitary lifestyle has been attributed to the combination of high association costs and low predation risk. Previous work revealed a positive correlation between association frequencies and habitat productivity, but did not measure the costs of association. In this comparative study, we show that females likely incur costs from involuntary, male-maintained associations, especially when they last for several days and particularly in the population characterized by lower association frequencies. Association maintenance therefore qualifies as another expression of sexual conflict in orangutans, and especially prolonged, male-maintained associations may qualify as an indirect form of sexual coercion. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00265-020-02948-4.

Morphological variants of silent bared-teeth displays have different social interaction outcomes in crested macaques (Macaca nigra).

OBJECTIVES: While it has been demonstrated that even subtle variation in human facial expressions can lead to significant changes in the meaning and function of expressions, relatively few studies have examined primate facial expressions using similarly objective and rigorous analysis. Construction of primate facial expression repertoires may, therefore, be oversimplified, with expressions often arbitrarily pooled and/or split into subjective pigeonholes. Our objective is to assess whether subtle variation in primate facial expressions is linked to variation in function, and hence to inform future attempts to quantify complexity of facial communication. MATERIALS AND METHODS: We used Macaque Facial Action Coding System, an anatomically based and hence more objective tool, to quantify "silent bared-teeth" (SBT) expressions produced by wild crested macaques engaging in spontaneous behavior, and utilized discriminant analysis and bootstrapping analysis to look for morphological differences between SBT produced in four different contexts, defined by the outcome of interactions: Affiliation, Copulation, Play, and Submission. RESULTS: We found that SBT produced in these contexts could be distinguished at significantly above-chance rates, indicating that the expressions produced in these four contexts differ morphologically. We identified the specific facial movements that were typically used in each context, and found that the variability and intensity of facial movements also varied between contexts. DISCUSSION: These results indicate that nonhuman primate facial expressions share the human characteristic of exhibiting meaningful subtle differences. Complexity of facial communication may not be accurately represented simply by building repertoires of distinct expressions, so further work should attempt to take this subtle variability into account.

Protamine and other proteins in sperm and seminal plasma as molecular markers of bull fertility.

Fertility is the most important aspect in the efforts to increase livestock populations. Protamine and various proteins in sperm and seminal plasma are the results of the molecular analysis which can be used as a marker of fertility. Each of the proteins plays an important role in the normal function of sperm, starting from the formation of sperm structure, motility, capacitation, cell protection, acrosome reactions, successful fertilization, egg activation, and embryonic development. Finally, these molecular components can be a marker of fertility and can help to diagnose the cases of infertility/subfertility in livestock in the field.

Relationship of frozen-thawed semen quality with the fertility rate after being distributed in the Brahman Cross Breeding Program.

BACKGROUND AND AIM: Various factors can reduce the quality of semen used for artificial insemination and have an impact on fertility decline, such as poor handling during frozen semen distribution. This study was aimed at assessing the quality of frozen-thawed semen after distribution in the field and its importance in maintaining fertility. MATERIALS AND METHODS: The Brahman Cross (BX) breeding program of PT Lembu Jantan Perkasa, Indonesia, was used. This program was preferred due to its adherence to guidelines that limit the effects of extraneous factors that may affect semen quality. Frozen-thawed semen samples from eight bulls with the same production code were analyzed and compared between the production site (artificial insemination [AI] center) and the field (BX breeding program). Total and progressive motility (PM) of sperm were determined using computer-assisted semen analysis. Plasma membrane integrity (PMI) was assessed using hypoosmotic swelling test, sperm viability using Eosin-Nigrosin staining, acrosome integrity using trypan blue-Giemsa staining, morphological abnormalities using William staining, and DNA fragmentation using toluidine blue staining. The fertility rate was determined using the conception rate (%) derived from AI data based on 502 AI services and 478 cows in the BX breeding program. A t-test was used to compare the quality of frozen-thawed semen before and after distribution. The relationship between the qualities of frozen semen after distribution in the field with fertility was analyzed using Pearson correlation. RESULTS: There was no significant difference (p>0.05) in the quality of frozen-thawed semen (sperm motility, PMI, viability, acrosome integrity, abnormalities, and DNA fragmentation) between the production site (AI center) and after distribution in the field (BX breeding program). The semen met the minimum standards for AI programs. Total motility (r=0.986), PM (r=0.961), sperm viability (r=0.971), PMI (r=0.986), and acrosome integrity (r=0.992) were all positively correlated (p<0.05) with fertility rate; while sperm abnormalities (r=-0.996) and sperm DNA fragmentation (r=0.975) were negatively correlated (p<0.05) with fertility rate. CONCLUSION: The study showed that to achieve the maximal and optimal fertility rate in bulls in an AI program, the overall quality of frozen-thawed semen in all aspects is critical. This can be achieved if the handling during distribution and storage, as well as the various factors that may affect the quality of semen in the field, can be controlled properly.

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle.

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.

Assessing female reproductive status of spectral tarsier (Tarsius tarsier) using fecal steroid hormone metabolite analysis.

The wild population of spectral tarsier is declining and attempts to breed the species in captivity have been of limited success. One possible reason for this is that information on the reproductive biology of Tarsius tarsier is extremely limited and data on the species reproductive physiology are completely lacking. We validated fecal estrogen (E-total) and progesterone metabolite (5-P-3OH) measurements for monitoring female ovarian activity and pregnancy. We used this approach to provide the first data on cycle and pregnancy length based on endocrine information in this species. We collected regular fecal samples in combination with observations on socio-sexual behaviors for a maximum of 15 months from three females maintained at Primate Research Center of Bogor Agricultural University, Indonesia. Hormonal profiles indicated that behavioral estrus was associated with marked elevations in fecal E-total concentrations followed by increases in 5-P-3OH levels indicating luteal function. Pregnancy was characterized by low levels of E-total and 5-P-3OH during the first month and markedly rising concentrations thereafter. An ovarian cycle length of 21.7 ± 5.7 days was found. Gestation length was 128d (live infant), 131d (stillbirth), and 164d (death of mother and infant due to dystocia). Despite the small sample size, the study demonstrates the overall validity of fecal sex hormone metabolite measurements for reproductive monitoring in female T. tarsier, as such, the methods described here may ultimately help to improve the breeding management of the species in captivity. They may also offer new opportunities for investigating basic questions of tarsier reproductive biology in the wild by using fecal hormone metabolite analysis to diagnose pregnant animals and determine reproductive rates in relation to ecological and other factors influencing tarsier reproduction. Thus, non-invasive assessment of female reproductive condition as described here may ultimately contribute to facilitate in and ex situ conservation efforts of this endangered primate species.

Characterization of Seminiferous Epithelium Stages in the Wild Javan Muntjac (Muntiacus muntjak muntjak) Using the Tubular Morphology Method.

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.

Sexual Behaviour of the Spectral Tarsier (Tarsius spectrum) in Captivity.

Tarsius spectrum is a primate species endemic to Sulawesi. Populations of the species have decreased due to habitat destruction and hunting. The sexual behaviour of T. bancanus and T. syrichta are known, but that of T. spectrum has not been reported until recently. The aim of this research was to study the sexual behaviour of T. spectrum in captivity. We observed 3 pairs of T. spectrum at the captive breeding facility of the IPB Primate Research Centre for 9 months using focal animal sampling. We showed that principal courtship behaviours were scent marking (36.7%) and genital marking for females (16.2%) and genital inspection for males (16.0%). Copulations lasted between 3 and 4 min, starting with the male mounting the female and thrusting quickly as many as 168-236 times followed by slow thrusting 9-20 times. When slow thrusting occurred, females vocalized up to 6 times. At the end of the copulation sequence, males remained motionless with their penis inserted within the female's genitalia for about 31 s. Copulation occurred only once for each pair during the observation period. Our results should be useful to support breeding programmes and conservation actions for tarsiers.

Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques.

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.