Age-dependent variations in proteomic characteristics of spermatozoa in Simmental bull.

Increasing the age of bulls results in a decrease in reproductive function, including a reduction in sperm quality, which plays a vital role in determining the fertility of bulls. Through a proteomic approach, this research aims to analyze the influence of age factors on various proteomes contained in bull sperm. Frozen semen samples from Simmental Bulls were categorized into three age groups: two, four, and ≥10 years old. Subsequently, the post-thaw sperm cells obtained were separated based on molecular weight using 1D-SDS-PAGE. Peptides extracted from the bands produced in each age group were subjected to LC-MS/MS analysis. A total of 72 protein types were identified, with 45 being detected in the 4-year-old group and 41 expressed in both the 2 and ≥10-year-old groups. The results provided insights into proteins' role in sperm metabolism across all age groups. Specifically, the 2-year-old group exhibited the expression of proteins associated with acrosome assembly and spermatid development (SPACA1). In contrast, those in the 4-year-old group were linked to motility (PEBP4) and sperm decapacitation factor (PEBP1). Proteins expressed in the 2 and -year-old groups were discovered to be involved in fertilization processes (TEX101). In contrast, the ≥10-year-old age group was associated with hyperactive movement related to capacitation (Tubulin). In conclusion, age influenced the differences observed in the proteomic profile of post-thaw Simmental bull sperm using the 1D-SDS-PAGE tandem LC-MS/MS approach.

Sperm HSP70: may not be an age-dependent gene but is associated with field fertility in Bali bulls (Bos sondaicus).

This study aimed to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls of different age groups and to examine its potential as a biomarker determining bull fertility. This study used frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years). Parameters of frozen semen quality analyzed included sperm motility and kinetics using computer-assisted semen analysis, sperm morphological defects using Diff-Quick staining, acrosome integrity using FITC-PNA staining, and DNA fragmentation using acridine orange staining. HSP70 gene expression characterization was analyzed using qRT-PCR, and HSP70 protein abundance was analyzed using enzyme immunoassays. Fertility field data were obtained by analyzing the percentage conception rate for each bull based on the artificial insemination service data contained in the Indonesian-integrated system of the National Animal Health Information System (iSIKHNAS). The results showed significant differences (P<0.05) in total and progressive motility, morphological defects of the neck and midpiece, and tail of sperm, and acrosome integrity between the age groups of Bali bulls. HSP70 gene expression and protein abundance showed no significant differences (P>0.05) in different age groups. HSP70 gene expression correlated with fertility rate (P<0.05). Age affected several semen quality parameters but did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule could be a biomarker that determines the fertility of Bali bulls.

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.

A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls.

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4-8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC-MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality.

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.

Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate.

Introduction: Freezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate. Methods: The classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method. Results: Bovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen-thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen-thawed sperm quality parameters. The overall quality of frozen-thawed sperm was closely related (p < 0.01) to the fertility rate. Conclusion: We may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen-thawed sperm quality in bulls.

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.

Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls.

OBJECTIVE: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. METHODS: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. RESULTS: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. CONCLUSION: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

Sperm Transcriptome Analysis Accurately Reveals Male Fertility Potential in Livestock.

Nowadays, selection of superior male candidates in livestock as a source of frozen semen based on sperm quality at the cellular level is not considered accurate enough for predicting the potential of male fertility. Sperm transcriptome analysis approaches, such as messenger RNA levels, have been shown to correlate with fertility rates. Using this technology in livestock growth has become the principal method, which can be widely applied to predict male fertility potential in the livestock industry through the analysis of the sperm transcriptome. It provides the gene expression to validate the function of sperm in spermatogenesis, fertilization, and embryo development, as the parameters of male fertility. This review proposes a transcriptomic analysis approach as a high-throughput method to predict the fertility potential of livestock more accurately in the future.

Implications of sperm heat shock protein 70-2 in bull fertility.

Heat shock protein 70 (HSP70) is one of the most abundant chaperone proteins. Their function is well documented in facilitating the protein synthesis, translocation, de novo folding, and ordering of multiprotein complexes. HSP70 in bovine consists of four genes: HSP70-1, HSP70-2, HSP70-3, and HSP70-4. HSP70-2 was found to be involved in fertility. Current knowledge implicates HSP70-2 in sperm quality, sperm capacitation, sperm-egg recognition, and fertilization essential for bull reproduction. HSP70-2 is also involved in the biological processes of spermatogenesis, as it protects cells from the effects of apoptosis and oxidative stress. Fertilization success is not only determined by the amount of sperm found in the female reproductive tract but also by the functional ability of the sperm. However, subfertility is more likely to be associated with changes in sperm molecular dynamics not detectable using conventional methods. As such, molecular analyses and omics methods have been developed to monitor crucial aspects of sperm molecular morphology that are important for sperm functions, which are the objectives of this review.

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.

Comparative analysis of various step-dilution techniques on the quality of frozen Limousin bull semen.

BACKGROUND AND AIM: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques. MATERIALS AND METHODS: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. RESULTS: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05). CONCLUSION: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.

Sperm protein markers for Holstein bull fertility at National Artificial Insemination Centers in Indonesia.

BACKGROUND AND AIM: Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels. MATERIALS AND METHODS: The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman's correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands. RESULTS: The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p<0.05). Five protein bands were found in the SDS-PAGE of sperm proteins of Holstein bulls with different concentrations. Sperm proteins with MWs of 17.51 kDa, 14.87 kDa, 33.71 kDa, and 41.97 kDa were abundant in the Holstein bulls with an HF level, while 55 kDa proteins were abundant in the LF level of Holstein bulls. The sperm of Holstein bulls in the HF level contained proteins of about 33.71 kDa that were not detected in the LF. CONCLUSION: The sperm protein with a molecular weight of 33.71 kDa was predicted to be a specific protein biomarker that influences bull fertility. Sperm fertilization abilities were also determined by the sperm proteins, the morphology of sperm acrosomes, and the quality of plasma membranes. This method can be used to select bulls with high fertility to increase the population of Holstein bulls.

Advances in Cryopreservation of Bull Sperm.

Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.

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.

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.

Sonogram of coccygeus muscle in dairy cows with different gestational ages.

BACKGROUND: The change in size and weight of the female reproductive organs during gestation and birth might be affect the perineal muscles and this condition in dairy cow not been reported. This study aimed to assess the ultrasonographic image of coccygeus muscle in 11 inseminated dairy cows with different gestational ages and postpartum. METHODS: Gestational age was calculated based on the record of artificial insemination and confirmed by using transrectal brightness mode ultrasonography. Perineal hair between the sacrum and ischium bones was shaved along 3-5 cm before being ultrasound. The images of perineal area were obtained by transcutaneous ultrasound using a 5.0 MHz transducer. The thickness and intensity of the coccygeus muscle were measured and analyzed by gestational status and postpartum to show the differences. RESULTS: The results showed that the thickness of coccygeus muscle increased with the increase in gestational age. Muscle intensity only increased at young gestational age. However, it decreased with the increase in gestational age (P < 0.05). CONCLUSIONS: The ultrasound image of coccygeus muscle was affected by gestational status, thus this method may be used as one of the new methods of indirect gestational detection on dairy cows.

Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans.

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.