The relationship of estrus character before double dose artificial insemination in Limousin Crossbred cow

Ongole cattle in smallholder farms on Java, Indonesia, have been crossed with Limousin, which is not controlled repeatedly. The character of estrus affects the success of Artificial Insemination. This study aimed to determine the relationship between estrus characters before double dose artificial insemination using frozen semen sexing. The research was conducted in Senggreng Village, Sumber Pucung District, Malang Regency. The material was 45 head of Limousin crossbred cow inseminated using sexing semen produced by the Singosari National Artificial Insemination Center (SNAIC). The sexed semen processed with the Percoll Density Gradient Centrifugation (PDGC) method. The average body value of cows between 3-5 (scale 1-9) with an average age of 1.8-7 years, normal reproduction, clear signs of estrus. The reproductive organs were examined by rectal palpation to confirm that the cows are not pregnant. Parameters of estrus character were vulvar color, cervical mucus, vaginal temperature, pH, vulvar swelling, and heat detector value. The result showed that the estrus characters, including vulva color, vulva swelling, cervical mucus, vaginal temperature, cervical mucus pH, and heat detector score in the first and second artificial insemination have a low relationship. The regression models produced can be used but are less precise to estimate the relationship between estrus characters because the coefficient of determination shows a low influence.

Relationship of motility and acrosome reaction on sexing sperm in Ongole Crossbred bull

Artificial insemination using sexing semen is expected to produce calves with the desired sex. One sexing sperm method is the percoll density gradient centrifugation method. This study aimed to determine the changes and the relationship between motility and acrosome reaction after sexing process using percoll density gradient centrifugation. The material used was semen of ±5 years old Ongole crossbred bull with a bodyweight of ±700 kg as many as three bulls with mass motility 2+ and individual motility 70%. The method used was to compare fresh semen with sexed semen after the cooling process. Parameters measured were motility characters using CASA analysis, which included motility parameters, progressive motility, capacitation, and no acrosome reaction. Statistical analysis used paired T-test to distinguish among fresh semen, after sexing and cooling process. In comparison, regression and correlation were used to analyze the relationship of capacitation and hyperactivation sperm with no acrosomal reaction with motility and progressive motility. The results showed that motility and progressive motility decreased after the sexing and cooling process. Meanwhile, the acrosomal reaction, capacitation, and hyperactivity increased.

Citrate-Mediated Acyl-CoA Synthesis Is Required for the Promotion of Growth and Triacylglycerol Production in Oleaginous Yeast Lipomyces starkeyi

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

Effect of Red Blood Cell Aging In Vivo on Their Aggregation Properties In Vitro: Measurements with Laser Tweezers

Red blood cell (RBC) aggregation highly influences hemorheology and blood microcirculation in the human body. The aggregation properties of RBCs can vary due to numerous factors, including RBC age. The aim of this work was to estimate in vitro the differences in the RBC aggregation properties of different RBC age populations in single-cell experiments using laser tweezers. RBCs from five healthy volunteers were separated into four subpopulations by Percoll density gradient centrifugation. Each subpopulation of the RBC was separately resuspended in autologous plasma or dextran 70 kDa (50 mg/mL). The aggregation force between the single cells was measured with holographic laser tweezers. The obtained data demonstrated an enhancement of RBC aggregation force in doublets with age: the older the cells, the higher the aggregation force. The obtained data revealed the differences between the aggregation and aggregability of RBC in dependence of the RBC in vivo age.

Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30–35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Kualitas Semen Beku Sexing Sapi Peranakan Ongole Menggunakan Volume Semen Awal Yang Berbeda

ABSTRAKInseminasi buatan dengan menggunakan semen sexing diharapkan menghasilkan pedet dengan jenis kelamin sesuai harapan. Tujuan dari penelitian ini adalah untuk mengetahui kualitas, proporsi, dan jumlah produksi straw sexing menggunakan metode sentrifugasi gradien densitas percoll dengan volume awal semen yang berbeda. Penelitian dilakukan di Loka Penelitian Sapi Potong, Kecamatan Grati, Kabupaten Pasuruan dan Laboratorium Reproduksi Ternak, Fakultas Peternakan Universitas Brawijaya, Malang. Materi yang digunakan adalah semen sapi peranakan ongole berumur berkisar lima tahun dan bobot badan berkisar 700 kg sebanyak tiga ekor, motilitas massa ≥2+ dan motilitas individu ≥70%. Metode yang digunakan adalah eksperimental dengan tiga perlakuan volume awal saat sexing, yaitu 1 (P1); 1,5 (P2); dan 2 (P3) ml dengan ulangan 11 kali (ulangan berfungsi sebagai kelompok). Data dianalisa menggunakan Rancangan Acak Kelompok (RAK). Hasil penelitian menunjukkan bahwa perbedaan volume awal semen tidak berpengaruh (menurun) terhadap motilitas, viabilitas, abnormalitas, konsentrasi, total spermatozoa motil, recovery rate dan proporsi spermatozoa (P>0,05). Pengaruh yang sangat nyata (meningkat) terhadap jumlah produksi straw (P<0,01). Ulangan penelitian ini memberikan pengaruh yang sangat nyata (meningkat) terhadap kualitas (motilitas, konsentrasi, viabilitas, abnormalitas, total spermatozoa motil, RR, proporsi dan jumlah straw) dan proporsi spermatozoa X dan Y (P<0,01). Total spermatozoa motil setiap perlakuan telah memenuhi nilai harapan (10 juta/straw). Proporsi spermatozoa X dan Y telah memenuhi nilai harapan (80%:20%).Kata Kunci: kualitas, proporsi, semen beku sexing, strawABSTRACTArtificial insemination using sexing semen is expected to produce calves with the expected sex. The aim of this study was to determine the quality, proportion, and quantity of sexing semen production using the percoll density gradient centrifugation method with different initial semen volumes. The research was conducted at the Beef Cattle Research, Grati District, Pasuruan Regency, East Java Province, Indonesia, and the Animal Reproduction Laboratory, Faculty of Animal Science, University of Brawijaya, Malang, East Java Province, Indonesia. The material used was semen from three Ongole crossbred bull aged around five years and the bodyweight of around 700 kg, mass motility of ≥2+, and individual motility ≥70%. The method used was experimental with three initial volume treatments during sexing, namely 1 (P1); 1.5 (P2), and 2 (P3) ml with 11 replications (replications function as groups). The data were analyzed using a randomized block design (RBD). The results showed that the treatment of differences in initial semen volume did not affect motility, viability, abnormalities, concentration, total motile sperm, recovery rate, and proportion of sperm (P>0.05). On the other hand, the difference in the initial volume of semen had a very significant effect (increased) on the amount of frozen semen production (P<0.01). Repeated research also had a very significant effect (increased) on the semen quality (motility, concentration, viability, abnormality, total sperm motility, recovery rate proportion, and straw production) and the proportion of spermatozoa X and Y (P<0.01). The total motile sperm for each treatment had met the expected value (10 million/straw). Proportions of spermatozoa X and Y have met the expected value (80%: 20%).Keywords: proportion, quality, sexing frozen semen, straw.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called “targeting by timing.” Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC–MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of “glycosylation by timing” during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoeisis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called ‘targeting-by-timing’. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand how glycosylation evolves during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation and N- and O-glycans present in each compartment were analyzed by liquid chromatography and tandem mass spectrometry. We found abundant paucimannosidic N-glycans and lack of O-glycans in early-formed azurophil granules (AG), whereas later-formed specific and gelatinase granules (SG and GG) contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis-x and sialyl-Lewis-x epitopes. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of ‘glycosylation-by-timing’ during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.