Percoll Density Gradient Centrifugation Research Articles

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Conception Rate of Filial Friesian Holstein Cows After Being Inseminated Using Unsexed and Sexed Semen

This research aimed to increase the reproduction of dairy cows with artificial insemination (AI) in Filial Friesian Holstein cows using sexed semen. This research was conducted in Pandesari Village, Pujon District, Malang Regency, East Java. The 114 Filial Friesian Holstein cows were used in this research and divided into three Treatments: T1: 38 cows were inseminated using unsexed semen, T2: 38 cows were inseminated using albumin sedimentation sexed semen, and T3: 38 cows were inseminated using Percoll density gradient centrifugation (PDGC) sexed semen. The material was selected by purposive sampling with a minimum body condition score (BCS) specification of 2.5 (scale 1-5); the material had normal reproductive organs and showed signs of heat/estrus. The parameters of this study are the percentage of non-return rate (NRR) 1, NRR 2, and conception rate (CR). The data obtained were analyzed using descriptive analysis. The differences in NRR and CR between the unsexed sperm, sexed sperm with albumin sedimentation, and sexed sperm with PDGC were analyzed with the chi-square test and were considered significant at p < 0.05. The chi-square test was carried out to compare the observed values with the expected values. The results showed that the success of artificial insemination was greater by using albumin-sedimented sexed semen compared to unsexed semen or PDGC-sexed semen, with NRR values of 1 (95%), NRR values of 2 (87%) and CR values of 63%. The conception rate of artificial insemination using albumin-sedimented sexed semen was 63% greater than that of artificial insemination using unsexed semen and PDGC-sexed semen, which obtained the same value of 47%.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

Quality and Proportion of Spermatozoa in the Percoll Density Gradient Centrifugation Sexing Method at Different Gradients using AndroMed® Diluent

The utilization of sexing technology is an effort made to improve the efficiency of livestock farming, which was created to predict the sex of the calves born so that it can be adjusted to the objectives of the farm. This study aims to analyze the quality and proportion of sexed spermatozoa using the percoll density centrifugation method at different gradients with AndroMed® diluent. The research material used was fresh semen from Belgian Blue Bull aged six years with a body weight of 600 kg and fresh semen quality motility ≥70%. This research is a laboratory experiment with two treatments and ten replicates. The treatments in the study were T0: Ten Gradient density percoll + AndroMed® and T1: Five Gradient density percoll + AndroMed®. The research analysis used a dependent t-test. The statistical analysis showed a significant difference (P<0.05) in treating ten gradients with five gradients in each layer regarding individual motility, viability, abnormality, and total motile spermatozoa. At the same time, in concentration, there was no difference (P>0.05) in the treatment of 10 gradients with five gradients. The average results on individual motility, abnormality, concentration, and total motile spermatozoa showed treatment of 10 gradients better than treatment of 5 gradients in each layer. At the same time, the variable viability showed that the gradient treatment is better than the five-gradient treatment. Proportion of spermatozoa in the Upper Layer T0 X (23.4%), Y (76.8%) and T1 X (16.9%), Y (83.1%). The proportion of Spermatozoa in the Bottom Layer was T0 X (84.1%), Y (15.9%), and T1 X (83%), Y (17%), respectively. In conclusion, sexing spermatozoa with the Percoll 10 and 5 Gradient Density Centrifugation method can separate X and Y spermatozoa.

Quantification of X sperm by raman spectroscopy in percoll density gradient centrifuged buffalo semen

The present study was conducted to observe effect of percoll density gradient centrifugation of buffalo bull semen on quantity of X sperms. Ejaculates were collected by artificial vagina method. Semen with mass motility >+3 and progressive motility >70 % were selected for experiment. X sperm Enrichment of semen was done by discontinuous percoll density gradient centrifugation and three groups were formed ie Group 1 (3 layer 70%, 50% and 30%) Group 2 (7 layer 70%, 60%, 50%, 40%, 30%, 20% and 10%) Group 3 (7 layer 80%, 70%, 60%, 50%, 40%, 30% and 20%). Centrifugation of semen of three groups and control (fresh semen without gradients) was done. After centrifugation, the supernatant part was removed and the pellet of each group was used for X sperm enrichment assessment by Raman spectroscopy. Results revealed that X sperm enrichment was higher in the pellets of Group 2 followed Group 3, Group 1 and Control as Raman peaks on DNA specific bands corresponds to more number of x sperm were higher respectively.

NO production of granulocytes associated with antibacterial immune response in Tegillarca granosa

Nitric oxide (NO) is a signaling molecule and immune effector produced by the nitric oxide synthases (NOS), which involved to various physiological processes of animals. In marine bivalves, hemocytes play important roles in antimicrobial innate immune response. Although hemocyte-derived NO has been detected in several bivalves, the immune function of hemocyte-derived NO is not well understood. Here, we investigated the antibacterial response of hemocyte-derived NO in the blood clam Tegillarca granosa. Two types of hemocytes including erythrocytes and granulocytes were isolated by Percoll density gradient centrifugation, their NO production and TgNOS expression level were analyzed. The results showed that NO was mainly produced in granulocytes and almost no detected in erythrocytes. The granulocytes showed significantly higher NO level and TgNOS expression level than the erythrocytes. And the TgNOS expression level was significantly increased in granulocytes after Vibro parahemolyticus challenge. In addition, the NO donor sodium nitroprusside (SNP) significantly increased the NO production of hemocytes to kill pathogenic bacteria. In summary, the results revealed that granulocytes-derived NO play vital roles in the antimicrobial immune response of the blood clam.

A Novel, Efficient Method to Isolate Chicken Primordial Germ Cells from Embryonic Blood Using Cell Culture Inserts.

Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the SSEA-1 and DAZL proteins and retained the ability to migrate in vivo. Overall, PGCs separated using cell culture inserts/CEF adhesion method retain their stem cell characteristics and migration ability. PGCs also exhibit good proliferation efficiency, making them suitable for subsequent transgenic experiments or genetic resource preservation.

Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2–4 cell stage. The intracellular ROS concentrations were investigated using 2′, 7′-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.

Reevaluating Symbiotic Digestion in Cockroaches: Unveiling the Hindgut's Contribution to Digestion in Wood-Feeding Panesthiinae (Blaberidae).

Cockroaches of the subfamily Panesthiinae (family Blaberidae) are among the few major groups of insects feeding on decayed wood. Despite having independently evolved the ability to thrive on this recalcitrant and nitrogen-limited resource, they are among the least studied of all wood-feeding insect groups. In the pursuit of unraveling their unique digestive strategies, we explored cellulase and xylanase activity in the crop, midgut, and hindgut lumens of Panesthia angustipennis and Salganea taiwanensis. Employing Percoll density gradient centrifugation, we further fractionated luminal fluid to elucidate how the activities in the gut lumen are further partitioned. Our findings challenge conventional wisdom, underscoring the significant contribution of the hindgut, which accounts for approximately one-fifth of cellulase and xylanase activity. Particle-associated enzymes, potentially of bacterial origin, dominate hindgut digestion, akin to symbiotic strategies observed in select termites and passalid beetles. Our study sheds new light on the digestive prowess of panesthiine cockroaches, providing invaluable insights into the evolution of wood-feeding insects and their remarkable adaptability to challenging, nutrient-poor substrates.

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

Siliceous scales in the centrohelid heliozoan Raphidocystis contractilis facilitate settlement to the substratum

The centrohelid heliozoan Raphidocystis contractilis has hundreds of small scales on the surface of the cell body. To understand the biological functions of the scales, comparative examinations were conducted between wild-type and scale-deficient strains that has naturally lost scales after long-term cultivation. The scale-deficient strain exhibited decreased adhesion to the substratum and had a lower sedimentation rate in water than the wild-type strain, suggesting that the scale may have the ability to attach quickly and strongly to the substratum. Percoll density gradient centrifugation showed that the scale-deficient strain had a lower density than that of the wild-type strain. In the wild-type strain, more scaled cells were observed in the higher specific gravity fractions. During the long-term culture of cells, only the cells suspended in the upper area of the flask were transferred to fresh medium. By repeating this procedure, we may have selected only cells that did not possess normal scales. In the natural environment, centrohelid heliozoans are easily flushed away if they cannot adhere strongly to the bottom. These results suggest that they use scales to ensure effective adhesion to the substratum.

Effect of percoll density gradient centrifugation on semen quality of X- sperm enriched crossbred bull semen

The present study was conducted to observe the effect of percoll density gradient centrifugation on quality of semen. Ejaculates were collected by AV method from Sahiwal bulls. X-sperm enrichment was done by percoll density gradient method i.e. 7 layers (70-10%). Centrifugation was done at 750 g (22-24°C) for 15 min. The pellets obtained were diluted in EYC medium. Semen quality was evaluated in fresh semen (Control), in pellet of normal centrifugation (Group I), supernatant of centrifugation in percoll density gradient (Group II) and pellet of centrifugation in percoll density gradient (Group III). To assess the quality of enriched semen pH, mass motility, progressive motility, live spermatozoa %, abnormal spermatozoa %, HOST % and intact acrosome % were evaluated. Number of progressively motile sperms in pellet of X- enriched semen were non-significantly increased and significantly (P<0.05) decreased in supernatant. The abnormal spermatozoa (%) were decreased in G III as compared to G II Live spermatozoa (%) were increased in enriched semen (pellet). Number of Intact sperms decreased significantly (P<0.05) in supernatant of percoll density gradient centrifuged Sahiwal semen. HOST responsive sperms number was not affected after percoll density gradient centrifugation. Thus, the semen quality of X sperm enriched semen by percoll density gradient method (7 layer 70%) was not affected hence it can be used to increase female calves’ birth after A.I.

The Quality and Proportion of Spermatozoa X and Y in Sexed Frozen Semen Separated with Percoll Density Gradient Centrifugation Method on Friesian Holstein Bull

The Quality and Proportion of Spermatozoa X and Y in Sexed Frozen Semen Separated with Percoll Density Gradient Centrifugation Method on Friesian Holstein Bull

Phenotypic and functional characterization of two coelomocyte subsets in Apostichopus japonicus

The hemocytes of invertebrates are composed of different cell subsets with different morphologies and structures. Different cell subsets have different immune functions, which play an important role in innate immune response against pathogens. However, the understanding of the classification of Apostichopus japonicus coelomocytes and the molecular basis of immune function of different cell subsets is very limited. In this study, two coelomocyte subpopulations of A. japonicus were isolated by Percoll density gradient centrifugation. They were identified from their morphological and structural characteristics, namely, spherical cells with a size of 10–12 μm spherical in shape and a large number of small granules inside; lymphocyte-like cells with a size of 4–5 μm spherical or oval in shape, and 1–3 filopodia. Functionally, the phagocytic capacity and lysosomal activity in spherical cells were significantly greater than those in lymphocyte-like cells. The results suggest that spherical cells may play a more critical role in the immune responses. Meanwhile, transcriptome sequencing analysis was performed to further clarify the functional differences between the two cell subsets. The data indicated significantly different gene expression patterns in them. Spherical cells tend to participate in immune defense, whereas lymphocyte-like cells tend to participate in energy metabolism. In addition, lymphocyte-like cells may convert oxidative phosphorylation to glycolysis by changing the manner of energy metabolism to quickly adapt to the energy demand of external stimuli. Spherical cells may respond to LPS stimulation through phagocytosis, and their response time is slower than that of lymphocyte-like cells. The expression of genes involved in endocytosis, phagocytosis, and lysosomal and humoral immunity in spherical cells was significantly higher than that in lymphocyte-like cells. These data provide valuable information for understanding the molecular basis of cellular and humoral immunity in A. japonicus.

Using gel microdroplets to develop a simple high-throughput screening platform for oleaginous microorganisms

The oleaginous yeast Lipomyces starkeyi is expected to be a new lipid source since this microorganism is capable of accumulating more than 85% lipid per dry cell weight. For effective utilization of oleaginous yeast, mutants with improved lipid production compared to the wild-type have been screened by methods such as single-cell sorting and Percoll density gradient centrifugation. Because these methods need to reculture all mutated oleaginous yeasts together in a flask, it is difficult to evaluate the growth of each individual mutant. Thus, screening for the slow-growing mutants with high-throughput has never been performed by conventional methods. In this study, we developed a high-throughput method using gel microdroplets (GMD). With this method, the growth and lipid production of L. starkeyi can be evaluated simultaneously. L. starkeyi grew in GMD and the size of these microcolonies was evaluated by scattered light. Finally, a mutant with a 10-fold delay in growth compared to the wild-type was obtained. Analysis of genetic information in this mutant could reveal valuable information about critical genes involved in the growth of these microorganisms, which could then be utilized further.

Dissecting the Chloroplast Proteome of the Potato (Solanum Tuberosum L.) and Its Comparison with the Tuber Amyloplast Proteome.

The chloroplast, the energy organelle unique to plants and green algae, performs many functions, including photosynthesis and biosynthesis of metabolites. However, as the most critical tuber crop worldwide, the chloroplast proteome of potato (Solanum tuberosum) has not been explored. Here, we use Percoll density gradient centrifugation to isolate intact chloroplasts from leaves of potato cultivar E3 and establish a reference proteome map of potato chloroplast by bottom-up proteomics. A total of 1834 non-redundant proteins were identified in the chloroplast proteome, including 51 proteins encoded by the chloroplast genome. Extensive sequence-based localization prediction revealed over 62% of proteins to be chloroplast resident by at least one algorithm. Sixteen proteins were selected to evaluate the prediction result by transient fluorescence assay, which confirmed that 14 were distributed in distinct internal compartments of the chloroplast. In addition, we identified 136 phosphorylation sites in 61 proteins encoded by chloroplast proteome. Furthermore, we reconstruct the snapshots along starch metabolic pathways in the two different types of plastids by a comparative analysis between chloroplast and previously reported amyloplast proteomes. Altogether, our results establish a comprehensive proteome map with post-translationally modified sites of potato chloroplast, which would provide the theoretical principle for the research of the photosynthesis pathway and starch metabolism.

Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue

Objective: To establish an optimized method for the isolation and purification of astrocytes from the neural tissues of young and aged rats. Then, the morphological and functional differences of astrocytes between young and aged rats were compared to explore the functional changes of astrocytes after aging and its possible mechanism in the aging process. Methods: Young (2 months old) and aged (20 months old) SD rats were used. Astrocytes in brain and spinal cord tissue were purified by 50% - 35% percoll density gradient centrifugation. Each group of cells was set up with three duplicate wells. After 72 h of culture, Glial fibrillary acidic protein (GFAP) which was astrocyte specific marker were detected by immunofluorescence to evaluate the morphological characteristics. Cell senescence markers (p16 and p21) and β- Galactosidase were detected by qPCR and staining respectively. The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) and anti-inflammatory cytokines were detected by qPCR. Results: Using 50%-35% percoll gradient separation, astrocytes were obtained with large number, good activity and purity of more than 95%, which could be used in subsequent experiments. Compared with the astrocytes in the nerve tissue of young rats, the astrocytes in the nervous tissue of the aged rats had fewer protrusions and tended to be activated in cell morphology; the positive rate of β -galactosidase staining was increased significantly and the expressions of p16 and p21 were increased (P<0.01). The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) were increased (P<0.05), and the expression of anti-inflammatory cytokine (IL-10) was decreased (P<0.05) in astrocytes of the aged rats nervous tissue. Conclusion: The percoll gradient of 50% - 35% could be used as a method for separation, purification and primary culture of astrocytes. With the increase of age, astrocytes undergo cellular senescence, showing a pro-inflammatory phenotype, promoting inflammaging of the nervous system, which may be one of the mechanisms of nervous system aging and neurodegenerative diseases.

Functional characteristics of YAP-positive hepatocytes expression in an early stage of NASH with transcriptome sequence analysis

Objective: To analyze and compare the differentially expressed genes (DEGs) of Yes-associated protein (YAP)-positive and negative hepatocytes and further understand the preliminary functional characteristics of YAP-positive hepatocytes in an early mouse model of nonalcoholic steatohepatitis (NASH) with transcriptome sequence (RNA-Seq). Methods: C57BL/6 mice were fed with methionine-choline deficiency (MCD) diet for 2 weeks to establish an early NASH model, and the control group was fed with normal diet. Liver tissue was stained with hematoxylin-eosin (HE) and Sirius red, and the pathological score was recorded. The expression of YAP and P-YAP were determined by immunohistochemistry (IHC) in liver tissues. Primary hepatocytes with viability greater than 90% were isolated and purified by collagenase perfusion combined with Percoll density gradient centrifugation. YAP-positive and negative hepatocytes were assessed by YAP antibody, flow cytometry and RNA-Seq analyses. Sequencing results were screened by GO, KEGG and interaction network analysis methods. RT-PCR was used to verify the expression levels of YAP and some DEGs in liver tissue model group. Two samples mean was compared by independent samples t-test. Results: Compared with the control group, the HE-stained liver tissue of MCD-induced mice at 2 weeks showed steatosis (pathological score 1.07±0.21), accompanied by lobular inflammation (pathological score 1.13±0.32) and ballooned hepatocyte (pathological score 0.80) ±0.20). Sirius red staining showed non-significant liver fibrosis (pathological score 0.40±0.40). IHC showed partial YAP-positive hepatocytes expression in an early stage of NASH. RNA-Seq analysis showed that clean reads of YAP-positive and negative hepatocytes were 49 310 604 and 5 4820 036, respectively. Compared with YAP-negative hepatocytes, YAP-positive hepatocytes had differential expression of 5 565 genes, including 1 662 up-regulated genes and 3 903 down-regulated genes. GO analysis of up-regulated genes showed that the metabolic processes related to mitochondrial functions, such as purine nucleoside triphosphate and nucleoside triphosphate were significantly enriched in biological processes (BP), while down-regulated gene analysis showed that olfactory-related receptor were significantly enriched in BP. KEGG analysis showed that DEGs were enriched in 292 pathways, and oxidative phosphorylation (OXPHOS) pathway was significantly enriched in signaling pathway. RT-PCR validated that inflammatory factors (interleukin-1β, interleukin-6), YAP and its target genes (Cyr61, Ankrd1), and Cox5b and Sdhc genes were significantly up-regulated in the OXPHOS pathway, which was consistent with the sequencing results. In addition, eight key genes with interaction network analysis were predicted. Conclusion: Changes in hepatocyte metabolic levels may be associated with increased YAP activity in an early stage of NASH.

Successful Artificial Insemination using Sexed and Non Sexed Semen on Limousin Crossbred Cow

Background: Beef cattle fattening business requires male cattle because they grow faster so that by using sexed semen (Y sperm) it is expected to get a male calf. The purpose of this study was to determine the success of artificial insemination using non-sexed and sexed frozen semen using percoll density gradient centrifugation (PDGC) method. Methods: A total of 120 female Limousin Crossbred were used in this study. The cows were raised on smallholder farms aged between 3-9 years with a body condition score (BCS) between 3-7 (score 1-9). The treatment was T0: 60 cows were inseminated using non-sexed frozen semen while T1: 60 cows were inseminated using sexed Y frozen semen. The insemination was performed with double doses at the 2nd and 8th hours after oestrus and deep insemination. The parameter measured were NRR-1, NRR-2, conception rate and pregnancy rates. Determination of pregnancy was performed using rectal palpation and ultrasonography (USG). Result: The results showed that NRR-1 and NRR-2 at T0 were 47 (78.33%) and 42 (70%), while at T1 were 37 (61.67%) and 35 (58.33%), respectively. While conception rate at T0 and T1 was 22 (36%) and 18 (30%), pregnancy rate at T0 and T1 was 31 (56%) and 19 (31.67%) and silent heat at T0 and T1 was 24 (40%) and 26 (43.3%).

ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.