Las subpoblaciones de espermatozoides y su calidad en fracciones producidas por la centrifugación de una sola capa en muestras frescas y normospérmicas de esperma de cordero

Single layer centrifugation (SLC) technique has been developed to select the best sperm population in the ejaculate in order to increase the fertilization rates by artificial insemination or in vitro fertilization. Normospermic ram semen samples containing 800 and 3,000 × 106 sperms/ml (C800 and C3000, respectively) were processed by SLC. Three sperm fractions were separated in each sample following silica-coloidal sperm centrifugation and sperm yield, quality and subpopulations were analyzed in each one. In C800 group, the sperm recovery rate did not vary in any studied fraction, but when samples were highly concentrated (C3000) the top fraction (F1) contained significantly higher spermatozoa than bottom fraction (F3). Also, it was observed that F1 in C3000 had got a significantly higher percentage of spermatozoa (53.2 %) than in C800, while the quantity of spermatozoa recovered in fraction 2 was lower (25.2 % vs 45.4 %). Based on the sperm motility parameters, three sperm subpopulations were identified: SP1, low velocity spermatozoa showing no progressive movement (19.1 %); SP2, rapid and progressive spermatozoa (43.7 %); and SP3, rapid spermatozoa but non-linear movement (37.2 %). While SLC has been implemented for sperm separation in suboptimal and/or low concentrated sperm samples, this trial demonstrates that SLC is not efficient to separate different sperm populations in normospermic ram sperm samples containing high concentrations of spermatozoa.

Effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine

Background and Aim: Studies have shown that the pH of the vagina during the course of fertilization may influence the migration of X- and Y-bearing spermatozoa and thus leading to skewness in the sex of the offspring. Hence, this study was carried out to check the effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine (Bos indicus). Materials and Methods: To check the effect of pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine, we used buffers of various pH ranging from 5.5 to 9.0 for swim-up procedure of sperm sample and collected upper and bottom fraction from the same buffer and checked the abundance of X- and Y-bearing spermatozoa by droplet digital polymerase chain reaction using X- and Y-chromosome-specific DNA probe. Results: The abundance of X- and Y-bearing spermatozoa was not differed significantly in either of the fraction collected. Conclusion: Thus, it appears to be unlikely that an immediate impact of pH on sperm can be a solitary impact on the sex of offspring in bovine. Keywords: droplet digital polymerase chain reaction, spermatozoa, swim-up.

Sex sorting sperm of sumba ongole bulls by using snakehead fish (Channa striata) albumin extract

The objective of this research was to investigate the potential of snakehead albumin extract (channalbumin) for sorting X and Y sperm of Sumba Ongole (SO) and its characteristic. Semen was collected from three SO bulls using artificial vagina and the freeze dried channalbumin was extracted from snakehead fish. Channalbumin column was made with different concentration ratio of top and bottom fraction: 2%:4%; 3%:5%; 4%:6% respectively and BSA 5%:10% as control. Semen was put in top fraction then incubated for 30 min at room temperature then each fraction was centrifuged at 1800 rpm for 10 minutes. The pellet was evaluated for motility, abnormality, viability, membrane integrity and head sperm morphometric. The results showed that the channalbumin capable to maintain sperm motility in the top fraction better than the bottom fraction. Sperm viability and membrane integrity in control group were significantly higher (P<0.05) than all channalbumin treatment. BSA 5%:10% has highest proportion of X and Y sperm (69%:76.77%) compared with 2%:4% (42.33%:79.13%), 3%:5% (55.97%:75.73%) and 4%:6% of channalbumin (62.77%:68%). It’s concluded that channalbumin 4%: 6% was effective for separation of XY sperm with higher proportion.

NEW UNIVERSAL METHOD FOR DETERMINATION OF ANTICOAGULANTS IN RODENTICIDES

Solid paraffin briquettes are one of the most modern types of rodenticide baits, because they protect it from the environmental exposure and do not reduce palatability of rodents. This paper proposes a simple universal method for the determination of brodifacoum, bromadiolone and difenacoum in solid briquettes, which are a mixture of paraffin, poison and filler, which can be used as grain, flour and the like. The difficulty in the analysis of baits containing paraffin, is to get rid of it. For this, we propose to use hexane-acetonitrile extraction in the system. Paraffin dissolves in hexane, while brodifacoum, bromadiolone and difenacoum do not. The resulting two-phase system was separated using a separatory funnel and the bottom fraction was collected. If any fillers were present in the sample, they were filtered until the mixture was separated, the residue was washed with acetonitrile. Before introducing the sample into the chromatograph, it was filtered twice with PTFE filters with a porosity of 0.45 µm. The best separation was achieved using a Thermo Acclaim® Surfactant column using acetonitrile and 0.1 M aqueous ammonium acetate solution (pH 5.4) as the mobile phase in a gradient elution mode. All three poisons were detected at a wavelength of 264 nm. This method has a sensitivity of 0.028 mg. The linearity range is from 0.00067 to 0.01%. The recovery rate for bromadiolone is 94%, for brodifacoum, 98%, and for difenacoum, 90%.

PENGARUH WAKTU INKUBASI PADA PROSES SEXING SPERMA BERBASIS GLUTATHIONE TERHADAP MOTILITAS DAN MEMBRAN PLASMA UTUH CHILLED SEMEN DOMBA LOKAL

The aim of this study was to examine the effect of incubation time on sexing process based on glutathione to the motility and Intact Plasma Membrane (IPM) of chilled ram semen. The separation of X and Y sperm conducted by using BSA method. This research was experimental research using completely randomized design (CRD) with three treatment namely, 45 minutes (T1), 60 minutes (T2) and 75 minutes (T3). Data were analyzed using Anova followed by Duncan's multiple range test. Material used in this research was the fresh semen from ram with sperm motility ≥ 70% and 5 mM of glutathione. The result of this research showed that the highest percentage of motility in upper and bottom fraction belong to T1 (69,7% dan 68,8%), followed by T2 (66,4% dan 64,5%) and T3 (57,9% dan 57,6%). In addition, the result of this research that the highest percentage of IPM in upper and bottom fraction belong to T1(75% dan 71,3%), followed by T2 (69,9% dan 68,4%) and T3 (66,9% dan 65,5%). It can be concluded that the incubation time of 45 minute is the optimum time on sexing process based on glutathione so that the motility and IPM of chilled ram semen can be maintained.

THE PROPORTION OF X AND Y SPERM, VIABILITY AND MOTILITY OF RAM SPERMATOZOA AFTER SEPARATED WITH WHITE EGG ALBUMIN

ABSTRACT The aim of this research was to determine the proportion, viability and motility of X and Y ram spermatozoa separated with egg white albumin. Sperm samples derived from Garut ram, which was collected by using an artificial vagina. Observations were made on spermatozoa fraction above and below each medium fraction treatment. There are treatment egg white albumin as separation medium, each medium consisting of fractions top and bottom fraction with different concentration: 1) P0 = sperma before separation (control); 2) P1 = 10% above fraction + 30% lower fraction; P2 = 25% + 45%; P3 = 25% + 75%. Data proportion of X and Y, viability and motility were analyzed statistically by Completely Randomized Design patern in the direction followed by Duncan’s Multiple Range Test for data with a real difference. Separation with egg white albumin affect significantly increased the proportion of spermatozoa X and Y (P≤0.05), but tends to decrease the viability and motility of spermatozoa.The proportion of spermatozoa X and Y was highest in treatment P3,76.76% of spermatozoa X (fraction above 25%) and 79.81% spermatozoa Y (75% lower fraction), with an average viability obtained respectively 68,9% (fraction above) and 59,7% (bottom fraction), motility 77,5% (fraction above) dan 84,0% (bottom fraction). It was concluded that the egg white albumin is very effective in changing the proportions of X and Y ram sperm with the quality of spermatozoa after separation feasible for applications insemination or processed into frozen semen. (Keywords: Garut ram, White egg albumin, Spermatozoa X and Y)

Mesostructure Evolution during Storage of Rubber Films from Washed Cream Latex and Skim Latex

Fresh field latex (FL) of Hevea brasiliensis was centrifuged to separate the major compartments: (i) the cream, (ii) the skim plus the C-serum, and (iii) the bottom fraction. Cream was dispersed in distilled water to prepare cream washed latex or CL sample. Skim was collected together with C-serum to prepare the skim latex or SKL sample. Then, films were made from FL, CL and SKL. The mesostructure (macromolecular structure and aggregates or gel) of the natural rubber films was studied by size exclusion chromatography coupled to a multiangular light scattering detector (SEC-MALS). The mesostructure was analyzed on fresh films and on films stored in the laboratory for three months (slow structuring). This study showed that FL and CL films had bimodal elution profiles of long and short polyisoprene chains as revealed by the two peaks on the chromatograms at low and high elution volumes, respectively. On the contrary, SKL films exhibited unimodal elution profiles at low elution volume and a highly abnormal molar masses elution profiles. After 3 months storage, the mesostructure of FL evolved dramatically especially the number-average molar masses (Mn), which increased by 60%. The mesostructure of CL films evolved at a lesser extent and, the mesostructure of SKL films exhibited no significant change. These results indicated (i) the skim rubber particles are not prone to slow structuring during storage in presence of serum and (ii) some constituents either in the serum or/and in the bottom fraction could still be present with cream and be involved in the slow structuring of cream particles latex.

Composition of Color Substances of Hevea Brasiliensis Natural Rubber

It is accepted that NR gives naturally occurring color, which restrict many applications such as light-color products. Therefore, characterization of color substances presenting in NR is very useful to develop the certain methodology to eliminate them completely or partly from NR in the future. In this work, an attempt was made to purify and characterize the color substances extracted from various fractions of Hevea rubber latex by certain methods, using high-resolution structural characterization techniques. It was found that the content of color substances extracted from fresh latex (FL), rubber cream, bottom fraction (BF), Frey Wyssling (FW) particles and STR 20 were different. Based on the high-resolution spectroscopic analyzes, it was found that the color substances extracted from NR were composed of carotenoids, tocotrienol esters, fatty alcohol esters, tocotrienols, unsaturated fatty acids, fatty alcohols, diglyceride and monoglyceride. The results will be useful for rubber-technologist to identify the origin to make obnoxious color in natural rubber, especially in some applications which are restricted by such the color.

Optimization of β-Quantification Methods for High-Throughput Applications

Background: Risk of cardiovascular disease is assessed, in part, by laboratory measurement of the concentrations of several lipoproteins. β-Quantification is a method of lipoprotein measurement that uses ultracentrifugation to partially separate lipoprotein classes. Although β-quantification is used largely in clinical and basic research, methods have not been described to allow the analysis of a large number of small-volume specimens with a short turnaround time. We report two variations of the traditional 5-mL method used by the Lipid Research Clinics Program that overcome these shortcomings. Methods: Two lower-volume modifications of the traditional 5-mL β-quantification method were developed. The methods used either 1 or 0.23 mL of specimen and required substantially less time for analysis (20 and 6 h, respectively) than the 5-mL method (2.5 days). The goal was to develop ultracentrifugation methods such that the concentration of cholesterol in the bottom fraction, from which LDL-cholesterol concentration is calculated, agreed with the 5-mL method. Fresh serum specimens (n = 45) were analyzed by the three methods to determine comparability of the methods based on the recovery of cholesterol in the bottom fraction after ultracentrifugation. To evaluate intrarun precision, replicate specimens (n = 17) were analyzed in a single run for each method. This experiment also evaluated how quickly the fractions would remix after separation by ultracentrifugation. For the 1-mL method, accuracy of the measurement of LDL- and HDL-cholesterol concentrations and the interrun precision were established by analysis of frozen serum specimens provided by the CDC, which established target values for the pools using reference methods. Results: No clinically significant differences in cholesterol concentrations in the bottom fraction were observed for the 1- and 0.23-mL methods, which had mean biases of 0.8% and 1.5% relative to the 5-mL method, respectively. Intra- and interrun variability was acceptable for each method, e.g., &lt;1.8% for cholesterol in the bottom fraction. Ultracentrifuged specimens were stable for at least 4 h with no evidence of contamination of cholesterol in the bottom fraction. For comparison specimens provided by the CDC, the 1-mL method met the accuracy and precision goals of the National Cholesterol Education Program for the measurement of HDL- and LDL-cholesterol concentrations (goals: total error &lt;13% and &lt;12%, respectively), with total errors of 6.45% and 5.43%, respectively. Conclusions: Both the 1- and 0.23-mL β-quantification methods are suitable substitutes for the traditional 5-mL method for use in clinical and basic research for the determination of LDL-cholesterol concentration. Both methods provide much higher throughput and require substantially less specimen volume. The 0.23-mL method can be performed in 1 day, but it is slightly less precise than the 1-mL method. In our laboratory setting, as many as 80 specimens are routinely processed per day using the 1-mL method.