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.

Profiling of seminal plasma proteins to identify the fertility of Simmental bull with low semen quality.

Objective: The present study analyzed the seminal plasma proteome and possible relationships between proteins and semen quality in azoospermic and normal Simmental bulls. Materials and Methods: Fresh semen plasma samples from the Lembang Artificial Insemination Center were used for this study, including one bull (76´ ejaculate) with very poor semen quality/azoospermia (poor fresh semen/infertile; PFS) and three bulls with normal semen quality (normal fresh semen; NFS) for proteomic analysis using a pooled system (NFS-Stud) (60´ ejaculate). The only males obtained with very low quality or azoospermia (PFS) had sperm motility of <10% (one head). Bulls with azoospermic conditions produce fresh semen without sperm or with very little sperm concentration. A total of 109 proteins were identified in the seminal plasma of Simmental bulls analyzed using liquid chromatography-mass spectrometry. Bioinformatics analysis was used to explore total protein, expression, function, and protein mechanism in the seminal plasma of Simmental bulls. Results: The results showed that the seminal plasma proteins expressed in NFS bulls include ELSPBP1, SIL1, HSPA13, angiotensin-1 covering enzyme, and CRISP1. On the other hand, B2M, C3, CFB, venin-2, and cathepsin S contribute significantly to PFS. The NFS bull proteins play important roles in sperm capacitation, protein transport, sperm motility, spermatogenesis, immune tolerance, and fertilization, while the PFS proteins perform apoptotic and antigen pathway functions. Conclusion: There is an interaction between proteins in the seminal plasma of males with poor semen quality (PFS) and cases of infertility (azoospermia) that cause a decrease in sperm quality in PFS 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.

Protein Identification of Seminal Plasma in Bali Bull (Bos javanicus).

The purpose of this study was to identify seminal plasma proteins in Bali bull and their potential as biomarkers of fertility. Semen was collected from 10 bulls aged 5-10 years using an artificial vagina. Fresh semen was then centrifuged (3000× g for 30 min). The supernatant was put into straws and stored in liquid nitrogen. The semen plasma protein concentration was determined using the Bradford method, and the protein was characterized using 1D-SDS-PAGE. Coomassie Brilliant Blue (CBB) was used to color the gel, and the molecular weight of the protein was determined using PM2700. A total of 94 proteins were identified in the seminal plasma of Bali bulls analyzed using LC-MS/MS (liquid chromatography-mass spectrometry). Proteins spermadhesin 1 (SPADH1), C-type natriuretic peptide (NPPC), clusterin (CLU), apoliprotein A-II (APOA2), inositol-3-phosphate synthase 1 (ISYNA1), and sulfhydryl oxidase 1 (QSOX1) were identified as important for fertility in Bos javanicus. These proteins may prove to be important biomarkers of fertility in Bali bulls. These proteins are important for reproductive function, which includes spermatozoa motility, capacitation, and acrosome reactions. This study provides new information about the protein content in seminal plasma in Bali bulls. The LC-MS/MS-based proteome approach that we applied in this study obtained 94 proteins. The identification of these seminal plasma proteins of Bali bulls and their potential as fertility biomarkers may have an impact on the success of future artificial insemination (AI).

Semen quality and frozen semen production in Pasundan bulls: A molecular weight perspective on seminal plasma and spermatozoa protein.

Objective: To determine the correlation between the molecular weight (MW) of proteins in seminal plasma and spermatozoa and the quality of fresh and frozen semen production in Pasundan bulls. Materials and methods: Nine selected Pasundan bulls, aged 5-10 years, from the Regional Artificial Insemination Center at Ciamis, West Java, Indonesia, were used in the study, with fresh semen sperm motility ≥70% and <70%. We analyzed the motility, viability, integrity of the intact plasma membrane (IPM), and the morphological characteristics of spermatozoa. 1D-SDS-PAGE analysis was performed to determine the protein profile by assessing MW, depicted as bands on the gel. Results: The motility, viability, and IPM of spermatozoa had lower values (p < 0.05) in Pasundan bulls named Bagaskara and Kertarajasa compared to the other bulls. Proteins with MW 35-50 kDa were not detected in the seminal plasma of Pasundan bulls, exhibiting low quality in fresh semen. The correlation analysis showed that the non-detected proteins with MW 35-50 kDa in seminal plasma correlated with spermatozoa motility (r = 0.421), viability (r = 0.424), and IPM (r = 0.428) so that fresh semen quality was low in both Pasundan bulls. Analysis of semen volume, spermatozoa concentration, and spermatozoa motility showed that the average frozen semen production of Pasundan bulls per ejaculate was 128.73 ± 15.35 straws. Conclusion: Protein analysis based on MW is a predictive indicator for the quality of fresh semen and the production of frozen semen in Pasundan bulls. Evaluation parameters of fresh semen quality by MW analysis can be used to select Pasundan bulls in Indonesia.

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.

Correlation between age, testosterone and adiponectin concentrations, and sperm abnormalities in Simmental bulls.

BACKGROUND AND AIM: Capacity for sperm production is affected by age, which is related to the morphology of sperm abnormalities and can affect fertility. The aim of this study was to evaluate the relationship between age and concentrations of testosterone and adiponectin with sperm abnormalities in Simmental bulls. MATERIALS AND METHODS: The study used 11 bulls, separated into three groups. The first group consisted of five bulls aged 4-5 years, and the second and third groups each consisted of three bulls, aged 6-7 and 8-10 years, respectively. The average sperm motility of the animals ranged from 57.66±2.60% to 70.17±0.22%. Blood samples were obtained from the coccygeal region of the animals. Testosterone and adiponectin analysis was performed using the enzyme-linked immunosorbent assay method. Sperm morphology was evaluated using carbol fuchsin-eosin staining according to the Williams method. Finally, correlations between testosterone and adiponectin concentrations, age, and sperm abnormalities were analyzed using Pearson's correlation analysis. RESULTS: The findings revealed a significant correlation (p<0.01) between the concentrations of testosterone and adiponectin (-0.538), age (-0.588), and abnormal sperm morphology (-0.912). Moreover, they revealed that the concentration of testosterone in the bulls aged 8-10 years was lower, at 21.89±4.56 ng/mL, compared to that in the bulls aged 4-5 years, at 36.15±1.29 ng/mL, and 6-7 years, at 35.16±5.39 ng/mL. The findings also revealed a positive correlation between adiponectin concentration and age (0.529) and sperm abnormalities (0.506). The increase in testosterone concentration was inversely related to the adiponectin concentration (-0.538). Moreover, the mean amount of abnormal sperm increased with increasing age: 3.82±0.33% in the group aged 4-5 years, and 4.40±0.72% and 10.20±1.97% in the groups aged 6-7 years and 8-10 years, respectively. CONCLUSION: The study data indicate that there is a decrease in testosterone concentration, a high adiponectin concentration, and an increase in abnormal sperm with increasing age in bulls.

Establishment of semen collection technique using electroejaculator and semen cryopreservation of Javan leopard (Panthera pardus melas Cuvier, 1809).

BACKGROUND AND AIM: The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to developelectroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen. MATERIALS AND METHODS: The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl®(Minitüb, Tiefenbach, Germany)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb, Tiefenbach, Germany) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation. RESULTS: The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL-1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%. CONCLUSION: This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology.

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.

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.