Semen collection in wild animals may be challenging for some methods such as electro ejaculator may cause pain and distress to the animals. Transrectal massage has been widely used in domestic and wild animals semen collection. Even though an artificial vagina may provide a condition that mimics the female reproductive tract and known to provide better results in some species, the application may face obstacles in the field. This study was conducted to evaluate the possibility of modified semen collection in banteng bull by combining the transrectal massage method followed by the use of an artificial vagina. The bull was habituated and trained for semen collection in chute under veterinarian supervision in Taman Safari Indonesia, Cisarua, Bogor, West Java. Semen samples from one Javan banteng bull were collected once a week. Banteng bull showed stud and penile protrusion during semen collection. Watery semen with volume ranged from 12 ml were successfully collected, with sperm progressive motility ranged from 25%-40%. Our study also showed sperm abnormalities comprising 18.11% abnormal sperm heads, 28.82% abnormal sperm tails and 0.94% teratoid form. The results demonstrated the possibility of this modified method as an alternative procedure for semen collection in banteng bull which may also be useful for other wild animals in field conditions

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 develop electroejaculator (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.

Abstract. Rosyada ZNA, Yoelinda VT, Kaiin EM, Gunawan M, Ulum MF, Tumbelaka LITA, Solihin DD, Agil M, Gunawan A, Purwantara B. 2023. Sperm osteopontin mRNA expression levels and its correlation on semen quality and fertility in Madura bulls. Biodiversitas 24: 563-570. Osteopontin (OPN) gene transcripts influence spermatogenesis and germ cell development. Therefore, transcriptome analysis is needed to identify the fertility factor OPN in Madura bull sperm. Madura cattle are crosses originating from Bos indicus (zebu) and Bos javanicus (banteng). This study aims to examine sperm osteopontin (OPN) mRNA expression levels and its correlation with semen quality and fertility in Madura bulls. Frozen semen samples from Madura bulls were categorized as high-fertile (n: 4, average field conception rate: 78.28±3.25%) or sub-fertile (n: 4, average field conception rate: 66.73±5.01%). In post-thaw semen samples, sperm motility, viability, membrane, acrosome, and DNA fragmentation index were evaluated. OPN expression in sperm total RNA was analyzed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). The data analysis between the two fertility groups was assessed with Student's t-test and Pearson Square. Receiver-operating characteristic analysis and diagnostic sensitivity and specificity calculations were performed. Sperm motility, acrosome intact, and DFI differ (p<0.05) between groups, whereas viability and membrane plasma intact have no significance (p>0.05). In high-fertile Madura bulls, OPN mRNA was upregulated (p<0.05). The OPN mRNA expression had a strong correlation with the field conception rate (R: 0.807, P<0.05), sperm motility, and intact acrosome (R: 0.899, p<0.01; R: 0.804, p<0.05) respectively. In contrast, OPN negatively correlated with sperm DFI (R: -0.764, p<0.05). The OPN predicted bull fertility with 81.3% accuracy, 75% sensitivity, and 75% specificity. Thus, OPN may serve as a potential biomarker of Madura bull fertility.

Abstract. Yoelinda VT, Arifiantini RI, Solihin DD, Agil M, Setiadi DR, Hastuti YT, Manansang J, Sajuthi D. 2023. Short Communication: Characteristics of the endangered Javan banteng (Bos javanicus) spermatozoa. Biodiversitas 24: 759-766. Morphology and morphometry of spermatozoa become essential fertility parameters in domestic and wild animals. Such morphological abnormalities can arise from genetic or environmental origins and cause fertility deprivation. Semen collection using transrectal massage has been used in several domestic and wild animals and is known to be simpler, safer, and less painful than artificial vaginas and electro-ejaculation. This study aimed to evaluate morphological and morphometrical features of the banteng spermatozoa head. The objective of this study was also, to evaluate the use of transrectal massage as a semen collection protocol. This study was conducted in Taman Safari Indonesia, Cisarua, Bogor, West Java, involving one banteng (Bos javanicus) bull. Semen samples were collected weekly, smeared, and stained with a modified Williams' staining method. This study found ten types of head morphological abnormality, comprising several major defects, with several types known to be the genetic origin. There were also nine types of spermatozoa tail abnormalities and teratoid form abnormalities. The total normal spermatozoa proportion in this study was 54.30±1.74%. Morphometric dimensions of length, width, and area head of banteng spermatozoa were 9.88±0.01 µm, 4.91 µm, and 38.12±0.05 µm2, respectively, with a tail length of 60.48±0.03 ?m. Although our result was limited by the number of samples available for the study, this first report on the banteng bull's sperm morphology and morphometry still need to be considered. The possibility of transrectal massage use for banteng semen collection protocol demonstrated in this study may be helpful to support the conservation approach in terms of breeding program strategy.

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.