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.

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.

Unsunnidhal L, et al. J Vet Sci. 2021 Aug;22(6):e76. https://doi.org/10.4142/jvs.2021.22.e76

The precursor protein of A‐kinase anchor protein 4 (proAKAP4), which is abundant in the fibrous sheath of sperm, plays an essential role in sperm motility and has been developed into one of the candidate molecular‐based fertility markers. This study aimed to identify proAKAP4 in the sperm of Indonesian dairy bulls and to investigate its correlation with various sperm quality characteristics. The findings are expected to be applied and developed to optimize the use of superior dairy bulls. Frozen semen from 20 Indonesian dairy bulls were used, which had previously been classified based on their fertility levels into two groups: high fertility (HF) and low fertility (LF). Analysis of frozen‐thawed sperm quality, such as progressive and total motility, sperm kinematics, membrane integrity, mitochondrial membrane potential, acrosome integrity, and capacitation status, was carried out in both groups. ProAKAP4 protein in frozen‐thawed sperm was analyzed using the Bull 4MID Kit with the ELISA technique. Each dairy bull showed varying proAKAP4 abundance ( p < 0.05) from one another. Frozen‐thawed sperm quality in HF bulls was higher ( p < 0.05) than in LF bulls, especially in total and progressive motility, sperm kinematics, membrane integrity, mitochondrial membrane potential, acrosome integrity, and noncapacitated sperm. The parameters of capacitated and acrosome‐reacted sperm showed the opposite results, where the quality in LF bulls was higher ( p < 0.05) than in HF bulls. The results of proAKAP4 identification showed that the protein abundance in HF bulls was higher ( p < 0.05) than in LF bulls. Correlation analysis confirmed a strong association ( p < 0.05) between proAKAP4 levels and sperm fertility rate. The proAKAP4 protein has great potential to be developed and applied as a marker for determining the fertility of dairy bulls and for selecting superior dairy bulls.

Background and Aim: The success in the handling and prevention of tuberculosis (TB) cases is highly dependent on their rapid detection, monitoring, and treatment. The efficacy of the Bacille Calmette–Guerin (BCG) vaccine is inconclusive in eastern Indonesia. The RV1980c gene of Mycobacterium tuberculosis encodes an antigenic protein that is considered to be a virulence factor, as it can stimulate the immune response in patients with TB. This study aimed to study the expression and epitope indicator of MPT64 recombinant proteins from clinical isolates of M. tuberculosis as immunoserodiagnostic candidates for pET SUMO plasmids from clinical isolates as candidates for serodiagnostic tests and recombinant vaccines. Materials and Methods: The polymerase chain reaction (PCR) product of the RV1980c gene was inserted into the SUMO pET plasmid, which was then transformed into Escherichia coli BL21 (DE3) cells and expressed in Luria Bertani media induced by 1.0 M IPTG. Subsequently, sequencing was performed and the results were analyzed using the ClustalW and National Center for Biotechnology Information BLAST software. The T-cell epitope prognosis was then explained by GENETYX version 8.0., for the prediction of B-cell epitope, as assessed using an Immune Epitope Database analysis. Results: The PCR product of the RV1980c gene had a length of 619 bp. Moreover, SDS–polyacrylamide gel electrophoresis and Western blotting revealed that the protein encoded by the Rv1980c gene weighed 36 kDa. We gained nine specific T-cell epitopes according to Iad Pattern position and eight epitopes according to Rothbard/Taylor Pattern Position; furthermore, we detected five B-cell epitopes in the RV1980c gene. Conclusion: The MPT64 protein encoded by the RV1980c gene carries epitopes that are realized by lymphocytes and represent potential immunoserodiagnostic candidates in diagnostic immunology.

Salah satu kendala dalam pelaksanaan kriopreservasi semen adalah tingginya kadar reactive oxygen species (ROS) yang dapat menyebabkan reduksi pada motilitas sperma dan viabilitas sperma post thawing. Strategi untuk mengatasi tingginya kadar ROS adalah dengan menambahkan antioksidan pada pengencer selama proses kriopreservasi. Tujuan dari penelitian ini adalah mengetahui pengaruh penambahan antioksidan selenium dan kurkumin dalam menurunkan stress oksidatif pada proses kriopreservasi semen sapi peranakan ongole (PO). Penelitian ini menggunakan semen yang dikoleksi dari sapi PO dengan vagina buatan. Sampel semen yang ditampung diperiksa secara mikroskopis dan makroskopis kemudian dibagi menjadi 4 perlakuan yaitu kontrol, penambahan selenium (sodium selenite) dengan konsentrasi 50 µM, kurkumin 10 µM dan kombinasi antara selenium 50 µM dengan kurkumin 10 µM. Semen yang telah dicampur dengan pengencer dan antioksidan kemudian dibekukan dalam proses kriopreservasi, selanjutnya parameter yang diamati meliputi pemeriksaan motilitas sperma, recovery rate dan viabilitas sperma. Semua parameter dianalisis statistika menggunakan One Way ANNOVA dan uji DMRT. Berdasarkan penelitian menunjukkan bahwa perlakuan kurkumin 10 µM memiliki nilai motilitas 48.00 ± 4.47%, recovery rate 66.66 ± 5.54% dan viabilitas 67.00 ± 4.30%, sedangkan perlakuan selenium 50 µM memiliki nilai presentase motilitas 46,00 ± 4,18%, recovery rate 63.90 ± 5.59% dan viabilitas 64.00 ± 3.93%. Pemanfaatan kombinasi kurkumin dan selenium tidak memberikan pengaruh yang nyata dibandingkan dengan kontrol. Kesimpulan penelitian ini adalah penambahan antioksidan kurkumin dan selenium bermanfaat dalam mempertahankan kualitas sperma sapi PO, namun perlakuan kombinasi kurang baik dalam mempertahankan kualitas sperma.