Addition Of Tert-butylhydroquinone Research Articles

Effects of Pre-Injection and Antioxidants in a Diesel Engine Fuelled with Methyl Esters of Waste Cooking Oil Biodiesel

Diesel engines are significant contributors to air pollution, particularly through emissions of nitrogen oxides (NOx), smoke, and carbon monoxide (CO). Finding sustainable fuel alternatives and additives to reduce emissions without compromising engine performance is imperative for environmental and public health concerns. This study investigates the impact of adding tert-butylhydroquinone (TBHQ) antioxidants to blends containing 20% Methyl Esters of Waste Cooking Oil (20MEOWCO) and 80% diesel fuel in Modified Common Rail Diesel (MCRD) engines. The experiment involves adjusting the pilot fuel injection timing to 36°CA bTDC (before Top Dead Centre) and the main injection timing to 15°CA bTDC, with a Nozzle Opening Pressure (NOP) of 500 bar. Biodiesel is produced from used cooking oil using standard procedures and then mixed with diesel fuel. Various concentrations of TBHQ are added to the 20MEOWCO fuel blend for the experiment. The findings indicate that introducing TBHQ in concentrations of 250 ppm and 500 ppm to the 20MEOWCO fuel blend results in a notable reduction of Oxides of Nitrogen (NOx) emission by 13% in MCRD engines. However, this reduction in emissions comes at the expense of increased specific fuel consumption, which is observed to rise by 2.1%. Furthermore, the study highlights a rise in smoke and carbon monoxide (CO) emissions by approximately 7–10% and 5-8%, respectively, under the experimental conditions. The results of this study suggest that the addition of TBHQ to 20MEOWCO blends holds promise for mitigating NOx emissions in MCRD engines.

A ratiometric electrochemical sensing strategy based on the self-assembly of Co NC/CNT and methylene blue for effective detection of the food additive tert-butylhydroquinone

It is crucial to achieve accurate and rapid detection of tert-butylhydroquinone (TBHQ) in the field of food safety, for the excessive addition of TBHQ in food is harmful to human health and evil to the environment and aquatic life. Therefore, researchers have done a lot of work on signal amplification through nanomaterials to achieve TBHQ detection, but the conventional single-signal detection strategy results in limited accuracy. In this work, an innovative and facile ratiometric electrochemical sensor for TBHQ detection was built based on advanced nanomaterial complexes carbon nanotube-encapsulated Co/nitrogen-doped carbon (Co NC/CNT) and selected internal reference signal methylene blue (MB) enhancing the accuracy by offering effective self-calibration. A linear relationship between the net peak current ratio between TBHQ and MB (ΔI (TBHQ)/ΔI (MB)) and the TBHQ concentration was obtained under the optimal experimental conditions, with two linear ranges of 0.1–20 μM and 20–100 μM and a limit of detection (LOD) of 0.054 μM (S/N = 3). Benefiting from the synergistic effects between Co NC and CNT and the ratiometric sensing strategy, the as-designed sensor for TBHQ detection showcased excellent selectivity, repeatability, reproducibility, stability, and satisfactory applicability in real edible oil samples.

Stability evaluation of biodiesel supplemented with synthetic and bio-based antioxidants by a pressurized accelerated oxidation method

This work examines pressurized accelerated oxidation by a RapidOxy tester as an alternative method for determination of biodiesel oxidation stability. Sunflower oil-based biodiesel was synthesized and treated with antioxidants: tert-butylhydroquinone (TBHQ) - a synthetic antioxidant known for its powerful protective effect, and a mixture of bio-based antioxidant compounds extracted from vinery waste, VWEeth. The antioxidant potency of TBHQ was evaluated at varying temperatures (110 - 140 ?C) and concentrations (250 - 2,000 mg dm-3) by the RapidOxy method; assessment of selected results was performed by comparison with relevant data obtained by the standard Rancimat method. VWEeth was added in two high dosages to biodiesel (87,500 and 150,000 mg dm-3) and analyzed at 140?C by the RapidOxy method. Both antioxidants at all applied dosages showed beneficial effects on improving the oxidative stability of biodiesel, but not all of the achieved improvements reached the stability minimum identified by the EN14214 standard. The lowest addition of TBHQ seemed to have a similar effect as the tested dosages of VWEeth but these additions did not increase the induction period above the limit of 8 h; two-fold higher quantity of TBHQ was successful in this respect, increasing the initial oxidation stability by a factor of about 2, which was determined by both methods. The RapidOxy method proved to be a very fast method suitable for testing a large number of samples, which is particularly important for efficient testing of different types and doses of antioxidants.

Distribution of 2-tert-butyl-1,4-benzoquinone and its precursor, tert-butylhydroquinone, in typical edible oils and oleaginous foods marketed in Hangzhou City, China

This study aimed to obtain a reliable evaluation about addition of tert-butylhydroquinone (TBHQ), and distribution of TBHQ and 2-tert-butyl-1,4-benzoquinone (TBBQ) contents in typical edible oils and oleaginous foods marketed in Hangzhou City. Briefly, the probability of labeled with addition of TBHQ in foods decreased from 36.45 ± 2.6% to 28.78 ± 3.7% in the period from 2018 to 2020. In the 135 analyzed samples, TBHQ contents were far less than the maximum legal additive amount, and TBBQ contents ranged from below its limit of quantification (LOQ) to 13.54 ± 1.15 mg/kg. The conversion rate from TBHQ to TBBQ in edible oils was 2.94 ± 1.17%, much lower than that in other food categories. Further research determined that the process method and food composition were the main factors for different conversion rates from TBHQ to TBBQ in various food categories. In addition, oil consumption was found to be the primary source of dietary intake of TBHQ and TBBQ.

Investigation of electrical properties of eco-friendly vegetable oil blended with bioactive compound.

Oxidative stability of prototypical groundnut oil (GO) and mustard oil (MO) blended with a significant characteristic addition of antioxidants, α-tocopherol (α-T) and TBHQ (tert-butyl hydroquinone), at different concentrations was analysed. α-T (natural) and TBHQ (synthetic) antioxidants at a different concentrations from 0 to 20 mg/L were blended with GO and MO to prepare 18 samples. Compositional analysis of groundnut oil and mustard oil was carried out using gas chromatography and mass spectra (GCMS). Electrical properties like dielectric constant, tanδ, specific resistance etc. were investigated at frequencies from 1 Hz to 10 MHz for the modified samples. Thermal properties like breakdown voltage, flash and fire points and viscosity of all samples were also premeditated. It was observed that dielectric constant (ε') of GO reduces from 3.58 to 2.82 with the addition of α-T but increases to 3.29 with the addition of TBHQ, whereas in MO, dielectric constant increases from 2.93 to 3.38 with the addition of α-T and rises to 3.27 with the addition of TBHQ at 15 mg/L. The synergistic effect of antioxidants in regulating the insulation nature of the oil is found to be more effective with α-tocopherol compared to TBHQ in GO. Higher breakdown voltage and more stability were observed in oil with the addition of antioxidant (TBHQ) in low concentration. The study would be useful in the selection of eco-friendly coolants in engineering and industry.

Influence of Lactobacillus plantarum on managing lipolysis and flavor generation of Staphylococcus xylosus and Saccharomyces cerevisiae in fish paste

To investigate the influence of Lactobacillus plantarum on managing lipolysis and flavor generation of Staphylococcus xylosus and Saccharomyces cerevisiae, three strains, Lactobacillus plantarum 120 (Lp-120), Staphylococcus xylosus 135 (Sx-135) and Saccharomyces cerevisiae 31 (Sc-31), isolated from traditional fermented fish were cocultured in fish paste with and without the addition of tert-butylhydroquinone (TBHQ). For comparison, the groups without inoculation of Lp-120 were acidified by glucono-delta-lactone (GDL) to exclude the effect of acidogenesis by Lp-120. The results showed that Sx-31 exhibited the highest liberation of FFAs at 4911 mg/kg dry matter (DM), followed by Sx-135 (4661 mg/kg DM) under monoculture and acidic conditions. However, when co-inoculated with Lp-120, although the growth of Sc-31 was slightly promoted, its lipolytic activity declined dramatically as evidenced by the lowest FFA concentration of 3414 mg/kg DM, accompanied by decreased free fatty acid (FFA) oxidation. Furthermore, when cocultured with Lp-120, the growth of Sx-135 was remarkably inhibited, and the release of FFAs (3984 mg/kg DM) also showed a moderate decline, but FFA oxidation was at the highest level. The results suggested that during fermentation of fish with the mixed-culture of the three strains, the liberation and oxidation of FFAs mainly resulted from Staphylococcus xylosus, followed by Saccharomyces cerevisiae. However, Lactobacillus plantarum inhibited the lipolysis and flavor generation caused by Staphylococcus xylosus and Saccharomyces cerevisiae, especially Saccharomyces cerevisiae.

Addition of tert-butylhydroquinone (TBHQ) to maize oil reduces lipid oxidation but does not prevent reductions in serum vitamin E in nursery pigs

BackgroundMaize oil is abundantly used in foods and feeds and is highly susceptible to oxidation. Consequently, commercially available antioxidants should be evaluated for effectiveness against lipid oxidation in swine diets. Our study was conducted to evaluate growth performance of nursery pigs fed oxidized maize oil and to determine effects of using antioxidants on oxidative status in a 2 × 2 factorial design. Two hundred eight weaned pigs were blocked by initial BW into 13 blocks, resulting in 4 pigs per pen and 13 pens per treatment. Dietary treatments included 6% unoxidized or oxidized maize oil, and 0 or 60 mg/kg of tert-butylhydroquinone (TBHQ), which was added after lipid oxidation. Data for growth performance were collected from 5 time periods of a two-phase feeding program (Phase 1 = d 0 to 12 and Phase 2 = d 13 to 34). Serum and liver samples were collected from one pig per pen, which had initial BW closest to average BW to determine oxidative status on d 34.ResultsOxidized maize oil was heated for 12 h at 185 °C with 12 L/min of air, yielding a peroxide value (PV) of 5.98 mEq O2/kg and TBARS of 0.11 mg MDA eq/g. Addition of TBHQ to diets containing oxidized maize oil decreased PV by 37% and increased the oil stability index by 69%. Final BW, ADG, ADFI, and G:F of pigs were not different among the four dietary treatments. However, pigs fed oxidized maize oil tended (P < 0.08) to increase hepatosomatic index by 5% compared with those fed unoxidized oil, and this was not affected by adding TBHQ. The serum vitamin E concentration of pigs fed oxidized maize oil was less (P < 0.03) than pigs fed unoxidized oil, but this reduction was not reversed by adding TBHQ. Finally, the serum and liver selenium concentration were not different among the treatments.ConclusionsThe addition of TBHQ did not affect growth performance and vitamin E status in pigs fed moderately oxidized maize oil, but TBHQ reduced lipid oxidation, enhanced the oil stability, and appeared to reduce oxidative stress.

Influence of Mineral Supplementation on the Results from Analysis of Flavonol Glycoside Content in Ginkgo biloba Dietary Supplements.

Flavonoids are a major component of Ginkgo biloba extract (GBE). Several studies have investigated chelate formation and the redox reaction between flavonoids and metal ions; however, the effect of mineral supplements on the results from the analysis of the flavonol glycoside content in products containing GBE dietary supplement remains unknown. In this study, the effects of commonly used mineral supplements on the recovery of quercetin from GBE-containing dietary supplements were investigated using conventional methods of flavonol glycoside determination. Mineral supplements containing Zn (II), Mn (II), and Fe (II) did not affect quercetin recovery, whereas Cu (II) and Fe (III) significantly reduced recovery (P<0.05). Quercetin oxidation was prevented by adding an antioxidant to the diluent (extraction solvent). Among the tested synthetic antioxidants, tert-butyl hydroquinone (TBHQ) promoted the greatest increase in quercetin recovery. The flavonol glycoside content of commercially available GBE-containing dietary supplements was analyzed using a conventional diluent or a diluent containing 20 mg/mL TBHQ. The amount of quercetin recovered from products containing Cu (II) was found to decrease with increasing hydrolysis duration and the duration in the final test solution state using the conventional diluent, while the TBHQ-containing diluent yielded consistent quercetin contents (P<0.05). These findings suggest that quercetin, a major aglycone of GBE flavonol glycosides, can be oxidized by Cu (II) and Fe (III) during the analytical process and, therefore, the total flavonol glycoside content may be underestimated. The addition of TBHQ to the diluent can improve the accuracy and reproducibility of flavonol glycoside content analysis in GBE-containing dietary products supplemented with minerals.

Simultaneous detection of reduced and oxidized forms of coenzyme Q10 in human cerebral spinal fluid as a potential marker of oxidative stress.

The redox balance of coenzyme Q10 in human plasma is a good marker of oxidative stress because the reduced form of coenzyme Q10 (ubiquinol-10) is very sensitive to oxidation and is quantitatively converted to its oxidized form (ubiquinone-10). Here we describe an HPLC method for simultaneous detection of ubiquinol-10 and ubiquinone-10 in human cerebral spinal fluid to meet a recent demand for measuring local oxidative stress. Since the levels of coenzyme Q10 in human cerebral spinal fluid are less than 1/500 of those in human plasma, cerebral spinal fluid extracted with 2-propanol requires concentration for electrochemical detection. Using human plasma diluted 500-fold with physiological saline as a pseudo-cerebral spinal fluid, we found that addition of tert-butylhydroquinone was effective in preventing the oxidation of ubiquinol-10. The optimized tert-butylhydroquinone concentration in the extraction solvent was 20 µM. The addition of 20 µM ascorbic acid or co-addition of tert-butylhydroquinone and ascorbic acid (20 µM each) were also effective in preventing the oxidation of ubiquinol-10, but ascorbic acid alone gave poor reproducibility. Good within day reproducibility was observed, and day-to-day analytical variance was excellent.

Effect of natural antioxidants on inhibition of parent and oxygenated polycyclic aromatic hydrocarbons in Chinese fried bread youtiao

The effects of three natural antioxidants (rosemary extract, tea polyphenol, and antioxidant of bamboo) on the levels of 16 polycyclic aromatic hydrocarbons (PAHs) and five oxygenated PAHs (OPAHs) in typical Chinese fried food youtiao were investigated, with synthesized antioxidant tert-butylhydroquinone (TBHQ) added to the frying oil for comparison. Soybean and palm oils were chosen as frying oils. The antioxidants were added to the frying oil in concentrations of 60, 120, and 180 mg/kg, and PAH and OPAH analysis was performed using GC-MS. Furthermore, the acid value, peroxide value, and polar components were detected to evaluate the antioxidant effect. Compared to samples without added antioxidants, the addition of TBHQ, rosemary extract, tea polyphenol, and antioxidants of bamboo reduced the total PAH and total OPAH concentrations by up to 30.30%, 23.47%, 11.38%, and 28.85%, respectively, for youtiao fried in soybean oil, and by up to 38.94%, 27.56%, 9.45%, and 39.26%, respectively, for youtiao fried in palm oil. The best inhibition was achieved by antioxidant of bamboo, which also showed good performance in slowing youtiao quality degradation. These results could inform good frying practice to improve the quality of fried food with low PAH and OPAH levels, and aid understanding of the new potential benefits of natural antioxidant extracts.

FTIR evaluation on the fuel stability of calophyllum inophyllum biodiesel: Influence of tert-butyl hydroquinone (TBHQ) antioxidant

‘Storage oxidation stability’ is one of the most significant criteria for appraising the biodiesel quality. Poor oxidation stability is the chief technical blockage related with the commercialization of biodiesel. The present paper investigates the effects of Tert- butyl hydroquinone (TBHQ) antioxidant additive concentrations on the long-term storage stability, thermal stability and accelerated oxidation stability of Calophyllum inophyllum biodiesel. Fourier transform infra-red (FTIR) spectroscopy was used to characterize the biodiesel oxidation variability, following the regions of C-H and O-H bonds in FTIR spectrum for different concentrations of TBHQ. Addition of TBHQ at 1000 ppm concentration (B100A3) with pure biodiesel enhances the oxidation stability by 94.67 %, storage stability by 14.47 % and thermal stability by 69.55 %; whereas further concentration of antioxidant deteriorates the formation of hydrophobic and hydrophilic clusters between biodiesel/antioxidant compounds, which is characterized by FTIR spectrum data. It is concluded that the Calophyllum inophyllum biodiesel could be accumulated for an extensive period by dosing 1000 ppm of TBHQ antioxidant.

TBHQ and peanut skin inhibit accumulation of PAHs and oxygenated PAHs in peanuts during frying

The effects of tert-butylhydroquinone (TBHQ) and peanut skins on the concentrations of polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (OPAHs) in fried peanuts were investigated. Sixteen PAHs and five OPAHs were quantified using gas chromatography-mass spectrometry (GC-MS). Addition of TBHQ to the frying oil reduced the total PAH and total OPAH concentrations by up to 71.75% and 74.80%, respectively. After frying, average PAH and OPAH levels were respectively 22.63% and 79.22% lower in peanuts with skins versus those without skins. The concentration of benzo[a]anthracene (BaA) was significantly correlated with that of benzo[a]anthracene-7,12-dine (BaAQ) in the fried peanuts. These results may be helpful for production of good quality fried peanuts with a low PAH content, as well as providing guidance for reducing PAHs in other fried foods.

Fuel properties of Brassica juncea oil methyl esters blended with ultra-low sulfur diesel fuel

Brassica juncea is a drought-tolerant member of the Brassicaceae plant family with high oil content and a short growing season that is tolerant of low quality soils. It was investigated as a feedstock for production of biodiesel along with evaluation of subsequent fuel properties, both neat and in blends with petroleum diesel fuel. These results were compared against relevant fuel standards such as ASTM D6751, EN 14214, ASTM D975, EN 590, and ASTM D7467. Crude B. juncea oil was extracted from unconditioned seeds utilizing a continuous tubular radial expeller. The oil was then chemically refined via degumming, neutralization and bleaching to render it amenable to direct homogeneous sodium methoxide-catalyzed transesterification. The principal fatty acid detected in B. juncea oil was erucic acid (44.1%). The resulting biodiesel yielded fuel properties compliant with the biodiesel standards with the exception of oxidative stability and kinematic viscosity in the case of EN 14214. Addition of tert-butylhydroquinone and blending with soybean oil-derived biodiesel ameliorated these deficiencies. The fuel properties of B5 and B20 blends of B. juncea oil methyl esters (BJME) in ultra-low sulfur (<15 ppm S) diesel (ULSD) fuel were within the ranges specified in the petrodiesel standards ASTM D975, EN 590 and ASTM D7467 with the exception of derived cetane number in the case of EN 590. This deficiency was attributed to the inherently low cetane number of the certification-grade ULSD, as it did not contain performance-enhancing additives. In summary, this study reports new fuel property data for BJME along with properties of B5 and B20 blends in ULSD. Such results will be useful for the development of B. juncea as an alternative source of biodiesel fuel.

Evaluation of the Oxidative Stability of Diacylglycerol‐Enriched Soybean Oil and Palm Olein Under Rancimat‐Accelerated Oxidation Conditions

AbstractThe oxidative stability of diacylglycerol (DAG)‐enriched soybean oil and palm olein produced by partial hydrolysis using phospholipase A1 (Lecitase Ultra) and molecular distillation was investigated at 110 °C by the Rancimat method with and without addition of synthetic antioxidants. Compared with triacylglycerol oils, the DAG‐enriched oils displayed lower oxidative stability due to a higher content of unsaturated fatty acids and a lower level of tocopherols. With the addition (50–200 mg/kg) of tert‐butylhydroquinone (TBHQ) or ascorbyl palmitate (AP), the oxidative stability indicated by induction period (IP) of these DAG‐enriched oils under the Rancimat conditions was improved. The IP of the diacylglycerol‐enriched soybean oil increased from 4.21 ± 0.09 to 12.64 ± 0.42 h when 200 mg/kg of TBHQ was added, whereas the IP of the diacylglycerol‐enriched palm olein increased from 5.35 ± 0.21 to 16.24 ± 0.55 h when the same level of AP was added. Addition of TBHQ, alone and in combination with AP resulted in a significant (p ≤ 0.05) increase in oxidative stability of diacylglycerol‐enriched soybean oil. AP had a positive synergistic effect when used with TBHQ.

Inactivation of Barotolerant Listeria monocytogenes in Sausage by Combination of High-Pressure Processing and Food-Grade Additives

Food-grade additives were used to enhance the efficacy of high-pressure processing (HPP) against barotolerant Listeria monocytogenes. Three strains of L. monocytogenes (Scott A, OSY-8578, and OSY-328) were compared for their sensitivity to HPP, nisin, tert-butylhydroquinone (TBHQ), and their combination. Inactivation of these strains was evaluated in 0.2M sodium phosphate buffer (pH 7.0) and commercially sterile sausage. A cell suspension of L. monocytogenes in buffer (109 CFU/ml) was treated with TBHQ at 100 ppm, nisin at 100 IU/ml, HPP at 400 MPa for 5 min, and combinations of these treatments. Populations of strains Scott A, OSY-8578, and OSY-328 decreased 3.9, 2.7, and 1.3 log with HPP alone and 6.4, 5.2, and 1.9 log with the HPP-TBHQ combination, respectively. Commercially sterile sausage was inoculated with the three L. monocytogenes strains (106 to 107 CFU/g) and treated with selected combinations of TBHQ (100 to 300 ppm), nisin (100 and 200 ppm), and HPP (600 MPa, 28°C, 5 min). Samples were enriched to detect the viability of the pathogen after the treatments. Most of the samples treated with nisin, TBHQ, or their combination were positive for L. monocytogenes. HPP alone resulted in a modest decrease in the number of positive samples. L. monocytogenes was not detected in any of the inoculated commercial sausage samples after treatment with HPP-TBHQ or HPP-TBHQ-nisin combinations. These results suggest that addition of TBHQ or TBHQ plus nisin to sausage followed by in-package pressurization is a promising method for producing Listeria-free ready-to-eat products.

Hydrogen peroxide- and cell-density-regulated expression of NADH-cytochrome b5 reductase in HeLa cells.

Environmental conditions regulate the expression of different antioxidant enzymes in cell culture. We have studied the effect of cell density and hydrogen peroxide on the expression of NADH-cytochrome b5 reductase in HeLa cells. Polypeptide levels of the NADH-cytochrome b5 reductase increased about three fold in confluent HeLa cells compared to sparse cells. Addition of H2O2 to HeLa cells altered expression levels of the NADH-cytochrome b5 reducatase in a concentration-dependent way, being sparse cells more sensitive to H2O2 addition than confluent cells. The presence of pyruvate, a H2O2 scavenger, produced a significant increment (200%) in the levels of NADH-cytochrome b5 reductase in sparse cells, but less increase (25%) in confluent cells, suggesting that generation of endogenous H2O2 could repress NADH-cytochrome b5 reductase expression, particularly in sparse cultures. Accordingly, confluent HeLa cells showed significantly lower levels of reactive oxygen species than cells in sparse cultures. Addition of tert-butylhydroquinone, a compound which generates reactive oxygen species through redox cycling, also reduced expression of the NADH-cytochrome b5 reductase. Increments in several antioxidant enzymes taking place during confluency could participate in the increase of NADH-cytochrome b5 reductase expression by reducing reactive oxygen species levels in cells. Overall, our results support that acute oxidative stress caused by H2O2 inhibits the expression levels of NADH-cytochrome b5 reductase, most likely due to inhibition of SP1 transcriptional activity. On the other hand, adaptation to H2O2 involved increased expression of the cytochrome b5 reductase, supporting the existence of additional regulatory mechanisms.

Response of human spermatozoa to an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone.

This study has investigated the effect of elevating intracellular calcium levels, using an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone (TBQ), on human sperm function. Isolated sperm samples from five fertile donors were incubated in a capacitating media for up to 6 hr. After 0, 3, and 6 hr incubation, sperm were exposed to a range of TBQ concentrations; 100 microM, 10 microM, and 1 microM, for a fixed incubation period of 5 min. Controls were run for each experiment where sperm were incubated for 5 min in the absence of TBQ. Sperm capacitation and the acrosome reaction were monitored prior to and after exposure to TBQ, using the Chlortetracycline assay. In addition, sperm motility was assessed at each time point and after sperm had been exposed to TBQ. The treatment of sperm with TBQ caused a significant increase in the number of capacitated sperm with an optimum response being achieved in the presence of 100 microM TBQ. However, sperm motility was found not to be effected by the addition of TBQ. The results from the present study suggest that elevating intracellular calcium levels in human sperm by short exposure to a high concentration of TBQ can rapidly accelerate the capacitation process. Furthermore, the observation that TBQ did not elicit a change in sperm motility suggests that TBQ may be highly specific in its mode of action by acting within the head region of human sperm.