Scale-based landmark for internal organs assessments of common carp (Cyprinus carpio) during digital radiography and ultrasonography imaging.

Medical imaging techniques such as digital radiography and ultrasonography are non-invasive and provide precise results for examining internal organs and structures within fish. Their effectiveness can be further enhanced by using body parts like scales as markers for the organs beneath them. This study utilized the number of scales as landmarks in digital radiography and ultrasonography to non-invasively evaluate the muscles, bones, and images of internal and reproductive organs of common carp (Cyprinus carpio). Digital radiography was performed in the dorsoventral and lateral views of the fish, whereas ultrasonography was conducted in longitudinal and transverse views on sequence scale numbers with brightness and colour Doppler-modes. Digital radiography of the common carp revealed the whole-body morphology, including the bony parts from the head, pectoral fins, dorsal fins, pelvic fins, anal fins, and vertebrae to the tail that appeared radiopaque. Internal organs were also observed, with the swim bladder and heart appeared radiolucent, while the intestines, liver, testes, and ovaries appeared radiopaque. Ultrasonography in brightness mode displayed the digestive organs, reproductive organs, and muscle thickness. Additionally, colour Doppler mode demonstrated blood flow within the heart's ventricle.

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.

Open AccessLack of association between coat color abnormalities in Bali cattle (Bos javanicus) and the coding regions of the MC1R and KIT genes.

Background and Aim: Coat color variations in cattle are known to be influenced by the melanocortin 1 receptor (MC1R) and receptor tyrosine kinase (KIT) genes. The presence of coat color abnormalities, such as white spots and albinism, in Bali cattle was the focus of this study. This study aimed to identify single nucleotide polymorphisms (SNPs) in the coding region of MC1R and exons 2 and 3 of KIT associated with coat color abnormalities in Bali cattle. Materials and Methods: The study included the analysis of 48 Bali cattle, including 20 individuals with standard coat color, 15 with white spots, and 13 with albinism. Total DNA was extracted using a DNA Extraction Kit, and MC1R (coding region) and KIT (exons 2 and 3) gene amplifications were analyzed using forward and reverse primers with polymerase chain reaction product lengths of 1071, 234, and 448 bp, respectively. The determination of MC1R and KIT gene diversity was analyzed through direct sequencing. Melanocortin 1 receptor and KIT gene sequence data were analyzed using BioEdit and MEGA6 to identify SNPs associated with standard and abnormal coat color phenotypes (white-spotted and albino) in Bali cattle. Results: No SNPs associated with coat color abnormalities were found in the coding region of MC1R and exons 2 and 3 of KIT genes in Bali cattle. However, the intron two regions of KIT contained the SNP g.70208534A>G, which showed a high degree of diversity. The AA genotype frequency was highest in albino Bali cattle, whereas the G allele frequency was highest and the A allele frequency was lowest in white-spotted Bali cattle. Conclusion: The results indicated that standard, white-spotted, and albino coat colors in Bali cattle could not be distinguished by analyzing the MC1R and KIT genes.

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.

Effect of supplementing L-cysteine and its group analogs on frozen semen quality of bulls: A meta-analysis.

Background and Aim: The quality of frozen bull sperm after thawing is influenced by the primary diluent and antioxidant. This meta-analysis was conducted to determine the effect of supplementing L-cysteine and its group analogs on the quality of frozen bull sperm. Materials and Methods: A total of 22 articles obtained from Google Scholar and Scopus were integrated into metadata. The effects of adding L-cysteine and its analogs (e.g., cysteine HCl and N-acetyl-L-cysteine), both of which are known as L-cysteine, were evaluated in this meta-analysis. The following parameters were examined: Abnormality, acrosome damage, acrosomal integrity, DNA damage, DNA integrity, malondialdehyde (MDA) content, plasma membrane integrity, pregnancy rate, progressive motility, sperm viability, and total motility. Data were analyzed using the mixed model methodology, with L-cysteine dosage as a fixed effect and different studies as random effects. Results: L-cysteine supplementation significantly increased the total motility (p < 0.05) and MDA content of semen, following a linear pattern. Progressive motility, acrosomal integrity, and plasma membrane integrity were significantly increased, showing a quadratic pattern (p < 0.05). Abnormality and acrosome damage were significantly decreased (p < 0.05), following a quadratic and linear pattern, respectively. Other parameters remained unaffected by L-cysteine supplementation. L-cysteine and cysteine HCl significantly inhibited (p = 0.001) acrosome damage in thawed frozen sperm compared with control sperm. Conclusion: Supplementing L-cysteine and its analog groups are recommended for freezing bull semen as it generally improves sperm quality.

Insight into the bioabsorption of Fe-based materials and their current developments in bone applications.

Iron (Fe) and Fe-based materials have been vigorously explored in orthopedic applications in the past decade mainly owing to their promising mechanical properties including high yield strength, elastic modulus and ductility. Nevertheless, their corrosion products and low corrosion kinetics are the major concerns that need to be improved further despite their appealing mechanical strengths. The current studies on porous Fe-based scaffolds show an improved corrosion rate but the in vitro biocompatibility is still problematic in general. Unlike the Mg implants, the biodegradation and bioabsorption of Fe-based implants are still not well described. This vague issue could implicate the development of Fe-based materials as potential medical implants as they have not reached the clinical trial stage yet. Thus, there is a need to understand in-depth the Fe corrosion behavior and its bioabsorption mechanism to facilitate the material design of Fe-based scaffolds and further improve its biocompatibility. This manuscript provides an important insight into the basic bioabsorption of the multi-ranged Fe-based corrosion products with a review of the latest progress on the corrosion & in vitro biocompatibility of porous Fe-based scaffolds together with the remaining challenges and the perspective on the future direction.

Postpartum perineal muscle sonogram in Madura beef cow.

BACKGROUND AND AIM: Ultrasonography (USG) is useful for non-invasively identifying changes that occur in soft tissue architecture. The objective of this research was to demonstrate postpartum (PP) uterine involution through the changes of perineal muscle intensity and thickness in Madura beef cow by ultrasonography. MATERIALS AND METHODS: Madura's breed cows used in the research consist of eight non-pregnant (NP) cows and three PP cow. The transrectal and transperineal USG imaging of NP cows was performed on days 1, 33, and 65. USG imaging of PP cows was performed every day starting from day 1 (24 h after parturition) until day 21 PP. Transrectal USG of the reproductive tract was performed for the cervix, corpus uteri, and cornua uteri. USG was performed transcutaneously over the perineal area for coccygeus and levator ani muscles at the longitudinal and transverse angles. Reproductive tract diameter and perineal muscle intensity and thickness were measured with ultrasound imaging. RESULTS: The analysis of the sonogram of PP cows showed that the diameter of the cervix, corpus uteri, and cornua uteri decreased within 21 days PP. The transverse view of the coccygeus muscle of PP cows showed decreased muscle intensity and thickness. On the other hand, the longitudinal view showed increased coccygeus muscle intensity and thickness. The transverse view of the coccygeus muscle of NP cows showed increased muscle intensity, while muscle thickness was reduced. Sonogram analysis of the levator ani muscle of PP cows showed decreased muscle intensity with increasing muscle thickness. However, imaging of the levator ani muscle of NP cows showed a decrease in both intensity and muscle thickness. There was a significant difference in the mean value intensity of the scanning view analysis results of the levator ani muscle of the PP cow (523.6 AU increased to 672.1 AU) and the NP cow (515.9 AU decreased to 465.4 AU). Furthermore, there was a significant difference (p<0.05) in the mean value thickness of both scanning view analyses of the coccygeus and levator ani muscles of PP cows (5.8 mm increased to 6.5 mm and 3.8 mm increased to 4.8 mm, respectively) and NP cows (8.8 mm increased to 9.1 mm and 5.9 mm decreased to 4.9 mm, respectively). CONCLUSION: We found that the perineal muscles, namely, the levator ani muscle and coccygeus muscle, change in size, and intensity during uterine involution as demonstrated on Madura beef cow.

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.

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.

EDTA-treated cotton-thread microfluidic device used for one-step whole blood plasma separation and assay.

This study aims to observe the wicking and separation characteristics of blood plasma in a cotton thread matrix functioning as a microfluidic thread-based analytical device (µTAD). We investigated several cotton thread treatment methods using ethylenediaminetetraacetic acid (EDTA) anticoagulant solution for wicking whole blood samples and separating its plasma. The blood of healthy Indonesian thin tailed sheep was used in this study to understand the properties of horizontal wicking and separation on the EDTA-treated µTAD. The wicking distance and blood cell separation from its plasma was observed for 120 s and documented using a digital phone camera. The results show that untreated cotton-threads stopped the blood wicking process on the µTAD. On the other hand, the deposition of EDTA anticoagulant followed by its drying on the thread at room temperature for 10 s provides the longest blood wicking with gradual blood plasma separation. Furthermore, the best results in terms of the longest wicking and the clearest on-thread separation boundary between blood cells and its plasma were obtained using the µTAD treated with EDTA deposition followed by 60 min drying at refrigerated temperature (2-8 °C). The separation length of blood plasma in the µTADs treated with dried-EDTA at both room and refrigerated temperatures was not statistically different (P > 0.05). This separation occurs through the synergy of three factors, cotton fiber, EDTA anticoagulant and blood platelets, which induce the formation of a fibrin-filter via a partial coagulation process in the EDTA-treated µTAD. An albumin assay was employed to demonstrate the efficiency of this plasma separation method during a one-step assay on the µTAD. Albumin in blood is an important biomarker for kidney and heart disease. The µTAD has a slightly better limit of detection (LOD) than conventional blood analysis, with an LOD of 114 mg L(-1) compared to 133 mg L(-1), respectively. However, the µTAD performed faster to get results after 3 min compared to 14 min for centrifuged analysis of sheep blood samples. In conclusion, on-thread dried-EDTA anticoagulant deposition was able to increase the wicking distance and has a better capability to separate blood plasma and is suitable for combining separation and the assay system in a single device.

The effect of hydrogen gas evolution of magnesium implant on the postimplantation mortality of rats.

BACKGROUND/OBJECTIVE: Hydrogen gas cavity is formed during in vivo degradation of magnesium implants. In many studies, the gas cavity is mostly punctured out subcutaneously. However, this procedure becomes inapplicable in certain internal surgeries; therefore, the effect of this gas cavity is worth further assessment. METHODS: In this study, we investigated the effect of hydrogen gas evolution on the mortality of rats and analysed the whole body capacity to relieve the gas. Porous pure-magnesium implants were implanted in the femoral bone defect of adult Sprague-Dawley rats up to 18 days, and their survival rate was calculated while the gas cavity size was measured, and its effect was analysed with support of radiographic and blood analysis. RESULTS: The gas cavity was rapidly formed surrounding the implantation site and obviously decreased the rats' survival rate. The gas was observed to swell the surrounding implantation site by filling the loose compartments and then dispersing subcutaneously to other areas. CONCLUSION: The rat's whole body capacity was unable to tolerate the rapid and persistent hydrogen gas cavity formation as shown by high postimplantation mortality.