Concentrations Of Butylated Hydroxytoluene Research Articles

Determination of butylated hydroxytoluene in edible oil using near-infrared spectroscopy combined with multivariate analysis

Edible oil is a fundamental component of human daily diet and has a wide impact on global agriculture and economy. The issue of additives in edible oil has always been a concern for its quality and safety, as excessive use of additives can be harmful to human health. This study suggests a quantitative detection technique for determining the concentration of butylated hydroxytoluene (BHT) in edible oil using near-infrared spectroscopy (NIRS) technology. Firstly, the spectral characteristics of edible oil samples with different concentrations of BHT are characterized using a near-infrared spectrometer. Then, particle swarm optimization (PSO), butterfly optimization algorithm (BOA), and fox optimizer (FOX) are employed to optimize the preprocessed spectral wavelengths. Finally, a detection model based on the optimized wavelengths is established using backpropagation neural network (BP-ANN) to achieve quantitative detection of BHT content in edible oil. The results show that the model based on the feature wavelengths optimized by the FOX algorithm has the best detection performance. This model takes 7 features as inputs, with a root mean square error of prediction (RMSEP) of 1.6116 g·kg−1 and a prediction determination coefficient (RP) of 0.9900 on the prediction set. The results demonstrate that near-infrared spectroscopy technology can accurately predict the BHT content in edible oil.

Spectrofluorimetric determination of butylated hydroxytoluene and butylated hydroxyanisole in their combined formulation: application to butylated hydroxyanisole residual analysis in milk and butter

Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are two antioxidants that have been extensively used in many applications. Both are well known for their debatable health risks due to their multiple intake sources. Therefore, conservative limits are set for them in different regulations adapted to the matrices in which they exist. Here we present a simple spectrofluorimetric method for the determination of BHT and BHA based on their native fluorescence and synchronous scanning mode. The type of solvent and the interval between emission and excitation wavelengths were carefully optimized. Under the optimized conditions, good linearities were obtained between the emission intensity and the corresponding concentrations of BHT and BHA over the range of 3–18 µg/mL and 0.1–7 µg/mL, respectively with a good correlation coefficient (r > 0.99). The limits of detection were 0.9 and 0.02 µg/mL, and the quantification limits were 3 and 0.05 µg/mL for BHT and BHA, respectively. The suggested procedure was validated according to ICH guidelines Q2 (R1). Furthermore, the method’s greenness was assessed by three different methods, and it proved to be eco-reasonable. The method was successfully applied to the determination of BHT and BHA in pharmaceutical formulations. We also applied the suggested method for monitoring the residual BHA in conventional, powdered milk and butter, with good recovery in spiked samples.

Butylated Hydroxytoluene (BHT) Improves the Post-Thaw Semen Quality in Low-Dose Sperm Cryopreservation in Murrah Buffalo Bull

BACKGROUND: Cryopreservation is an important technique for the long-term storage of semen for artificial insemination (AI). Buffalo spermatozoa are sensitive to cryopreservation procedures because of the presence of a high amount of polyunsaturated fatty acids in the plasma membrane. OBJECTIVE: To study the effect of different concentrations of BHT on the quality of Murrah buffalo bull semen for low-dose cryopreservation. MATERIAL AND METHODS: Semen was collected from four high fertile Murrah buffalo bulls (6 ejaculates each) using an artificial vagina. A total of 24 ejaculates were collected from each bull twice a week using an artificial vagina. Every sample was split into four parts: Control without additives; and three treatments with BHT at 0.5 mM, 1 mM or 2 mM. Semen was cryopreserved at low-dose sperm cryopreservation of 20, 15, 10 and 5 million sperm per aliquot after supplementation of BHT. Semen samples were evaluated for fresh, pre-freeze and post-thaw stages. RESULTS:There was a significant increase (p<0.05) in sperm quality parameters, such as progressive motility (%), viability (%), HOST response (%), acrosome integrity (%) and post-thaw motility, with the addition of 0.5-1 mM BHT. CONCLUSION: The addition of BHT in Murrah buffalo semen improves the low dose cryopreservation quality in a dose-dependent manner.

Effect of a polymerization inhibitor on the chemomechanical properties and consistency of experimental resin composites.

This study investigated the effect of butylated hydroxytoluene (BHT) inhibitor on degree of conversion (DC), flexural strength (FS), flexural modulus (FM), Knoop microhardness (KH), microhardness reduction (HR), and consistency of experimental resin composites at different BHT concentrations: C0 (control-0%); C0.01 (0.01%); C0.025 (0.025%); C0.05 (0.05%); C0.1 (0.1%); and C0.5 (0.5%). For the consistency, the composites were tested immediately after being exposed to a dental chair headlight (0, 20, 40 and 60 s). Data concerning DC, FS, FM, KH, and HR were submitted to one-way ANOVA, while the consistency data was submitted to 2-way ANOVA; mean values were then compared (Tukey's test; α=0.05). The KH, FS and FM analyses showed no significant difference among the composites tested. For DC, C0 showed the highest mean value (74.2%) and differed only from C0.5 (67.2%). For HR, C0.5 showed the lowest mean (13.09%) value and differed from C0 (26.4%) and C0.01 (24.87). The consistency analysis showed no difference among C0.05, C0.1 and C0.5, considering 0 and 20 s of light exposure, while C0 (14.07 mm), C0.01 (13.97 mm), and C0.025 (14.18 mm) showed higher mean values at 0 s when compared to 20 s (12.67, 12.77 and 13.05 mm, respectivelly). Polymerization occurred within 40 s of light exposure for C0, C0.01, C0.025, and C0.05 and within 60 s for C0.1. In conclusion, the BHT concentrations had no significant influence on FS, FM and KH. The higher the BHT concentration, the longer was its handling time under light, with a significant improvement in the HR, but a decrease in DC. Therefore, BHT at 0.1% showed the best outcomes concerning all the BHT concentrations tested.

Study of Rapid HPLC Method for the Determination of Antioxidants in Foods, and Standard Solution Stability during Storage

In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.

Effectiveness of the Natural Antioxidant 2,4,4'-Trihydroxychalcone on the Oxidation of Sunflower Oil during Storage.

This study aimed to investigate the effectiveness of 2,4,4′-trihydroxychalcone as a natural antioxidant on the oxidation of sunflower oil during an 88-day storage period and to compare its strength with the synthetic antioxidant butylated hydroxytoluene (BHT). Seven groups of the sunflower oil samples were prepared: pure oil (control), oil treated with different concentrations (100, 500, and 1000 ppm) of 2,4,4′-trihydroxychalcone, and oil treated with different concentrations (100, 500, and 1000 ppm) of BHT. Specific parameters, namely, the peroxide value (PV), acid value (AV), p-anisidine value (p-AnV), thiobarbituric acid reactive substance (TBARS) value and total oxidation (TOTOX) value were used to assess the extent of the deterioration of the oil by estimating the primary and secondary oxidation products. The results showed that 2,4,4′-trihydroxychalcone effectively decreased the production of the primary and secondary oxidation products of sunflower oil during storage, as indicated by reductions in the PVs, AVs, p-AnVs, TBARS values and TOTOX values of the sunflower oil. When compared to BHT, 2,4,4′-trihydroxychalcone showed either a similar or stronger effect in inhibiting the primary and secondary oxidation products. These findings suggest that, 2,4,4′-trihydroxychalcone is a suitable natural alternative to synthetic antioxidants to improve the oxidative stability of sunflower oil.

Migration analysis, antioxidant, and mechanical characterization of polypropylene-based active food packaging films loaded with BHA, BHT, and TBHQ.

Polypropylene (PP) based active composite films were prepared by adding butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylated hydroquinone (TBHQ) antioxidants using the extrusion molding process. All concentrations of BHT, 2% to 3% BHA, and 3% TBHQ significantly increased the tensile strength (TS) of the composite films compared with control films. Increasing antioxidant concentration decreased TS values for BHT films, whereas an opposite trend was observed for BHA and TBHQ films. BHA at <2%, BHT at >2%, and TBHQ at all added concentrations significantly reduced elongation at break (Eb ) of the composite films compared to control films. Water vapor permeability (WVP) of 1% BHT film was not significantly different from control. However, other antioxidants especially at increased concentrations significantly increased WVP values. TBHQ films with 300% to 662% increase had the highest WVP and BHT films with 5% to 81% increase had the lowest WVP among composite films. All three antioxidants had a negative effect on the transparency of the films; however the effect of BHA at higher concentrations was greater. The antioxidants did not change the color attributes of the films. Films containing all antioxidants showed 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, which increased with increase in their concentration, especially for those containing 3wt.% BHT and TBHQ. Overall, incorporating BHA and BHT into a PP matrix improved mechanical, barrier, antioxidant properties, and film appearance and consequently were proposed for the development of antioxidant active PP films. TBHQ film is not recommended for food packaging because of its weak mechanical properties (lower Eb and TS values, higher WVP, and greater migration).

Synergistic Effect for a Mixture of 3-Butyl-5-Amino-6-Methyluracil and Butylated Hydroxytoluene during Inhibited Styrene Autoxidation

The antioxidant effect of a mixture of 3-butyl-5-amino-6-methyluracil and butylated hydroxytoluene (BHT) was studied in a model system of initiated radical chain oxidation of styrene in chlorobenzene at 310 K. The maximum synergistic effect was observed at a ratio of ~0.75 between uracil derivative and BHT concentrations. The mechanism of synergistic action was discussed.

Microbubble ozonation of the antioxidant butylated hydroxytoluene: Degradation kinetics and toxicity reduction

Butylated hydroxytoluene (BHT) is recognized as a crucial pollutant in aquatic environments, but efforts to achieve its complete removal are without success. The aim of this study was to investigate the degradation efficiency of BHT in water using ozone microbubbles (OMB), coupled with toxicity change assessment at sub-lethal BHT concentrations (0.34, 0.45 and 0.90 μM) based on oxidative stress biomarkers in Daphnia magna. The efficiency of OMB on ozone gas mass transfer was assessed and the contribution of hydroxyl radicals (·OH) in the degradation of BHT was determined using p-chlorobenzoic acid (pCBA) probe compound and a ·OH radical scavenger (sodium carbonate, Na2CO3). The ozone gas mass transfer coefficient (kLa = 1.02 × 10−2 s−1) was much larger than the ozone self-decomposition rate (kd = 8 × 10−4 s−1) implying little influence of self-decomposing ozone in the volumetric ozone transfer during OMB generation. Generally, OMB improved ozone gas mass transfer (1.3–19-fold) relative to conventional ozone techniques, while indirect reaction of BHT with ·OH was dominant (82%) over the direct reaction with molecular ozone. Addition of 15, 25 and 35 mM Na2CO3 reduced BHT degradation by 30, 50 and 65%, respectively, indicating the significance of ·OH in the degradation of BHT. Increase in initial BHT concentration correspondingly reduced its removal rate by OMB possibly due to increase in metabolites produced during ozonation. Post BHT treatment exposure tests recorded significant (p < 0.05) reductions in oxidative stress (according to enzyme activities changes) in D. magna compared to pretreatment tests, demonstrating the effectiveness of OMB in detoxification of BHT. Overall, the results of the study indicate that OMB is extremely efficient in complete degradation of BHT in water and, consequently, significantly (p < 0.05) reducing its toxicity.

Butylated hydroxytoluene protects bull sperm surface protein-P25b in different extenders following cryopreservation.

Aim:The aim of this study was to investigate the effects of different concentration of butylated hydroxytoluene (BHT) on sperm membrane surface protein “P25b” from cryopreserved bull semen in either lecithin based Bioxcell® (BX) or two egg-yolk based extenders, tris-egg yolk (TEY), and citrate-egg yolk (CEY).Materials and Methods:Forty-five semen samples, 15 each were extended with either BX, TEY, or CEY extender which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0, and 3.0 mM/mL) of BHT. The extended semen samples were frozen at a concentration of 20×106/mL in 0.25 mL straws and stored in liquid nitrogen for 2weeks. The frozen samples were thereafter thawed, proteins extracted and analyzed for quantities of protein P25b through direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel densitometry. Peptides were confirmed by Western blotting (WB).Results:Results showed that supplementation of BHT improved (p<0.05) quantity of protein P25b at concentrations of 0.5mM/mL for BX and at 1.0 mM/mL for TEY and CE when compared with the controls and other treatments.Conclusion:BHT supplementation at 0.5 in BX and 1.0 mM/mL in TEY and CEY has protected bull sperm fertility marker protein P25b in frozen-thawed bull sperm.

Self-assembled lignin nanospheres with solid and hollow tunable structures

The fabrication of nanospheres with tailored structure has attracted much attention in material applications. Herein we found that the food additive, butylated hydroxytoluene (BHT), was able to adjust the solid and hollow structure of lignin nanospheres. At BHT concentrations greater than 0.3 mg/mL in tetrahydrofuran (THF), the obtained lignin nanospheres displayed a hollow structure due to phase separation between THF and water caused by BHT molecules, while the lignin nanospheres obtained without adding BHT exhibited a solid structure. BHT and lignin molecules containing a high content of the carboxy group did not participate in the formation of the nanospheres. Increases in the water stirring and dropping speeds reduced the diameter of the nanospheres, while increasing the initial concentration of lignin and BHT resulted in an increase in nanospheres diameters. Moreover, the shell wall thickness and single hole size could be controlled by employing the concentration of BHT and lignin. This work provides novel insights into the preparation of lignin nanospheres with tunable structures.

Effect of butylated hydroxytoluene supplementation on ultrastructure of crossbred bovine spermatozoa

The present study was planned to observe the effect of Butylated hydroxytoluene (BHT) on ultra structure changes in bovine crossbred spermatozoa. The sperm plasma membrane is the primary site of damage induced by cryopreservation. There are many harmful conditions such as cold shock, osmotic stress, ice crystal formation or oxidative damage etc which cause sperm cryo-injury and loss of sperm viability and fertility during cryopreservation. At the time of cryopreservation there were considerable ultra structure changes to the acrosomes and middle piece studied by transmission electron microscopy. Semen from two crossbred bulls were collected and extended with egg yolk tris dilutor along with addition of different concentrations of BHT i.e. 0.0mM (Control), 0.5mM (T 1 ) and 1.0mM (T 2 ), respectively. The effect of BHT on ultrastructural alterations was revealed by Transmission electron microscopy during post dilution (Stage I), post equilibration (Stage II) and post thaw (Stage III), respectively. TEM results of spermatozoa at Stage I, II and III showed that major ultrastrucural alterations occur in plasma membrane and outer acrosomal membrane. Group T 1 and T 2 showed significantly lower number of ultrastructural defects compared to control at Stage I, II and III. The study indicated that 1.0mM BHT increases the cryosurvival of sperm as revealed by post thaw lower ultrastructural defects of bovine bull spermatozoa

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.

ADIÇÃO DE HIDROXITOLUENO BUTILADO (BHT) NO DILUIDOR ACP-106c PARA CONGELAÇÃO DE SÊMEN CANINO

Resumo: Este estudo foi realizado para determinar o efeito de hidroxitolueno butilado (BHT) sobre a qualidade do sêmen canino congelado e descongelado, utilizando o diluidor à base de água de coco em pó (ACP-106c). Para tanto, foram realizadas quinze coletas de sêmen provenientes de cinco cães. O sêmen obtido foi diluído em ACP-106c acrescido de glicerol e gema de ovo. As amostras foram então transferidas para tubos contendo diferentes concentrações de BHT (0; 0,5; 1,0 e 2,0 mM). Em seguida, as amostras foram envasadas, congeladas e armazenadas em nitrogênio líquido. O sêmen coletado foi avaliado in natura quanto aos seguintes parâmetros: coloração, volume da fração espermática, motilidade total, vigor, concentração, morfologia e funcionalidade de membrana espermática. Após uma semana, as amostras foram descongeladas e avaliadas por meio de análise computadorizada, como também foram realizadas análises da funcionalidade de membrana e da morfologia espermática. A motilidade progressiva no grupo BHT 2,0 mM foi significativamente superior (P &lt; 0,05) do que a do grupo BHT 0 mM (27,6 ± 11,7% vs. 19,0 ± 9,5%, respectivamente). Em todos os demais parâmetros avaliados, não houve diferença entre os grupos testados. Portanto, conclui-se que a adição do BHT ao diluidor ACP-106c não afetou a qualidade do sêmen canino pós-descongelação.

Effect of seminal plasma and butylated hydroxytoluene (BHT) concentration on ram sperm freezability

The effect of removing seminal plasma (SP) by centrifugation before ram semen cryopreservation was assessed. Simultaneously, we studied the supplementation of different concentrations of BHT as antioxidant (0.6mM, 2mM and 5mM) on the extender in order to find an optimal concentration in enhancing a successful ram sperm cryopreservation. Therefore, fresh ejaculates from 8 rams of approximately 2 years old were collected, immediately mixed in equal quantities and split into 2 aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600×g for 10min, while the other aliquot was kept unwashed. Afterwards, washed and unwashed sperm were re-suspended in one of different TCG-based media with 5% glycerol and 15% powdered egg yolk (PEY) supplemented or not with BHT and/or SP according to the treatments: 1) washed sperm; 2) washed sperm+0.6mM BHT; 3) washed sperm+2mM BHT; 4) washed sperm+5mM BHT; 5) washed sperm+0.5mL of SP; 6) washed sperm+5mM BHT+0.5mL of SP; 7) unwashed sperm and 8) unwashed sperm+5mM BHT. Afterwards, sperm samples were refrigerated at 5° C for 4h before being packed in 0.25mL straws and frozen in nitrogen liquid vapors. After thawing, plasma and acrosomal membrane integrity and mitochondrial activity were determined by flow cytometry while sperm motility and morphometry by CASA (mean±SEM, n=6). Our results showed that seminal plasma removal enhanced sperm cryosurvival and the addition of BHT (5.0mM) improved most of the quality parameters of thawed washed and unwashed sperm, irrespective of the presence of SP in the extenders.

Exploring the mode of binding between food additive “butylated hydroxytoluene (BHT)” and human serum albumin: Spectroscopic as well as molecular docking study

Abstract Butylated hydroxytoluene (BHT) is a food additive and used as an antioxidant in food products. BHT also concerning several health risks such as cancer, asthma and behavioral disorder in children. Human serum albumin (HSA) is the main constituents of blood plasma and known as a carrier protein because it has a very strong ability to bind a variety of ligands. The binding of a drug or small molecules to serum albumin is a crucial determinant of its pharmacokinetic and pharmacodynamic profile. This study investigates the interaction between BHT and HSA at pH 7.4 using various spectroscopic techniques such as UV–visible absorption spectrophotometry, fluorescence quenching, circular dichroism (CD) and molecular docking. The interactions of BHT with human serum albumin in the UV–visible range of spectrum exhibits increased absorption peak at 280 nm that elevates with increasing concentrations of BHT. BHT was also found to quench the fluorescence of human serum albumin (HSA). However, the fluorescence quenching constant was decreased with increasing the temperature, indicating that the interaction between BHT and HSA takes place via the static quenching mechanism. The thermodynamic measurements indicated that both hydrogen and hydrophobic forces involved in the binding of BHT with HSA. Moreover, the negative ∆ G value showed that the binding process was spontaneous. Far-UV CD data suggested the marginal conformational changes in HSA due to BHT interactions. Furthermore, molecular docking study suggested that BHT interacted with HSA at domain III via hydrophobic interaction. Conclusively, spectroscopies, as well as computational analysis extend our understanding of BHT interaction with serum albumin, which has far reaching applications in the food industry.

Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage

In this research the antioxidant activity of water extracts of Bifurcaria bifurcata (BBE) at different dose against butylated hydroxytoluene (BHT) was evaluated in canola oil. Water extracts were firstly characterized in terms of total solid and polyphenolic compound contents, and their antioxidant activity together with that of BHT was evaluated using several in vitro tests (DPPH, ABTS, ORAC and FRAP). Next, the progress of lipid oxidation was assessed in canola oil added with five BBE concentrations (200, 400, 600, 800 and 1000ppm) and two BHT concentrations (50 and 200ppm) using an accelerated oxidation test. The progress in lipid oxidation was monitored by assessing some chemical indices (peroxide value, p-anisidine value, and conjugated dienes) during oil storage and some volatile compounds at the end of the storage period. BBE showed a significant antioxidant effect, being this ability concentration-dependent. The extent of lipid oxidation was inversely related to BBE dose, specially with regard to primary oxidation products. At the highest level of BBE, significant decreases of primary and secondary oxidation products, with respect to the control, were obtained with reduction percentages of 71.53%, 72.78%, 68.17% and 71.3% for peroxides, conjugated dienes, p-anisidine and TOTOX values, respectively. A level of 600ppm or higher concentration of the extract inhibits the lipid oxidation in a similar way than BHT at 200ppm. Regarding the inhibition of the formation of volatile compounds, both BBE and BHT strongly inhibited the formation of volatiles during oil storage, being this inhibition similar for all the concentrations of BBE and BHT essayed. Overall, results indicated that BBE can be used as a potential natural additive for improving oxidative stability of canola oil.

Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm

The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were frozen according to a standard protocol. After thawing of samples, sperm motility, viability, membrane integrity, total abnormality and lipid peroxidation were assessed. The greatest (P<0.05) values for total sperm motility, viability and plasma membrane functionality and least values for malonedialdehyde (MDA) concentration were observed in samples supplemented either with 1mM BHT or 2mM BHA. However, the progressive motility was greater (P<0.05) only in samples treated with 2mM BHA. In conclusion, the use of 1mM BHT or 2mM BHA in extender improves the freezing capacity of stallion sperm by reducing oxidative stress during freeze-thaw process.

Butylated hydroxytoluene can reduce oxidative stress and improve quality of frozen–thawed bull semen processed in lecithin and egg yolk based extenders

The aims of this study were to evaluate the effects of anti-oxidant butylated hydroxytoluene (BHT), when added at different concentrations into lecithin-based Bioxcell® (BX) and two egg-yolk-based; Tris (TY) and citrate (CE) semen extenders, on post-thaw bull sperm quality and oxidative stress. A total of 30 ejaculates from three bulls were collected using an electro ejaculator. Ejaculates were extended with one of the BX, TY and CE extenders, which contained different concentrations (0.0 – control, 0.5, 1.0, 1.5, 2.0 and 3.0mM/ml) of BHT. The extended semen samples were chilled to 4°C, and then frozen slowly to −196°C in 0.25ml straws before being stored in liquid nitrogen for 2 weeks. Results showed that supplementation of BHT improved (P<0.05) general motility, progressive motility, morphology, acrosome integrity, DNA integrity and malondialdehyde of sperm at 0.5mM/ml for BX and at 1–1.5mM/ml of BHT for TY and CE when compared with the control. However, greater concentrations of 2.0 and 3.0mM/ml of BHT had a detrimental (P<0.05) effect compared with the control with all extenders evaluated. In conclusion, BHT supplementation at lesser concentrations (0.5–1.5mM/ml) could improve frozen–thawed bull sperm quality by reducing oxidative stress produced during the freezing–thawing procedures in either lecithin or egg-yolk based extenders.

Effect of butylated hydroxy toluene on the quality of frozen goat semen

Ejaculates (40) collected from 5 Beetal bucks twice weekly were used to study the effect of 0 (control), 150, 200 and 300 μM butylated hydroxy toluene (BHT) on the quality of frozen semen by split sample technique. After removal of the seminal plasma, the semen was primarily extended with Tris extender (1: 5) considering the volume of semen prior to removal of seminal plasma and then split into four parts and finally extended with equal volume of Tris extender containing 0 (control), 300, 400 and 600 μM BHT. Consequent upon addition of equal volumes of Tris extender the rate of extension of semen rose to 1: 10 and eventual concentration of BHT in the extender was 0, 150, 200 and 300 μM. The extended semen was cooled gradually to 5°C @ 1°C/3 min and equilibrated in the cold handling cabinet for 4 h at 5°C. The extended semen was filled in French mini straws (0.25 ml) and polyvinyl alcohol powder of different colours were used for sealing the extended semen for identification of semen of different bucks processed with different concentrations of BHT. The freezing of semen was done by rapid horizontal vapour freezing technique using liquid nitrogen. The mean percent post thaw sperm motility, live sperm, live intact acrosome and HOST-reacted sperm were significantly higher in the extender with 200 μM BHT than with 150, 300 μM BHT and control. The release of ALT and AST from post thaw spermatozoa was also the lowest (33.75 ± 0.17 and 221.61 ± 0.73 U/L respectively) in the extender containing 200 μM BHT. Based on sperm motility, live sperm, live intact acrosome, HOST-reacted sperm, and ALT and AST release, 200 μM BHT in Tris extender was superior to 0, 150, and 300 μM BHT in freezing Beetal buck semen. It was concluded that BHT at 200 μM provided a better protection to the functional capacity of buck spermatozoa for cryopreservation.