Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm

1998 ◽  
Vol 10 (4) ◽  
pp. 347 ◽  
Author(s):  
L. G. Sánchez-Partida ◽  
B. P. Setchell ◽  
W. M. C. Maxwell
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Detrimental Effect ◽  
Compatible Solutes ◽  
Egg Yolk ◽  
Motile Sperm ◽  
Motility Of Sperm ◽  
Ram Sperm ◽  
Amino Acid Glycine

The effect of the compatible solutes proline, glycine betaine and trehalose in Tris-based diluents at varying pH, concentrations of egg yolk or glycerol on the post-thaw motility characteristics and fertility of ram sperm was examined. In addition, the amino acid glycine was compared with proline, glycine betaine and a standard Tris-based diluent. Post-thaw motility was assessed using a Hamilton–Thorn motility analyser. In the presence of glycerol and egg yolk, proline and glycine betaine improved the post-thaw motility characteristics of ram sperm. Regardless of the pH of the diluent at which semen was frozen, the percentage of motile sperm was higher when frozen in the presence of proline or glycine betaine than in their absence, whereas proline and glycine betaine only improved the progressive and rapid percentages of sperm for semen frozen in diluents at pH lower than 7.0. When semen was frozen in the absence of egg yolk or glycerol all the motility characteristics were reduced. Increasing the concentration of egg yolk in the diluent from 5% to 10, 15 or 20% had no effect on the post-thaw motility of sperm. The addition of 27 mM of proline or glycine betaine to the diluent also improved post-thaw motility. However, at a concentration of 81 mM, proline and glycine betaine had a detrimental effect on the percentage of motile sperm. Trehalose had no effect on the motility of sperm frozen in glycerol-containing diluents, but motility was lower after cryopreservation in glycine than in Tris-, proline- or glycine betaine-based diluents. There were no differences in the fertility of sperm frozen in Tris-, proline or glycine betaine diluents after cervical or laparo-scopic insemination of ewes.

scholarly journals Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Anirudha Dutta ◽  
Vijay Parashar ◽  
E. Fidelma Boyd
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Vibrio Parahaemolyticus ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Amino Acid Residues ◽  
Choline Transporter ◽  
Content Type ◽  
Highly Effective

ABSTRACT Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport. IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus. We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

scholarly journals Investigations of dimethylglycine (DMG), glycine betaine and ectoine uptake by a BCCT family transporter with broad substrate specificity in Vibrio species

2020 ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Anirudha Dutta ◽  
Vijay Parashar ◽  
E. Fidelma Boyd
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Vibrio Parahaemolyticus ◽  
Full Range ◽  
Turgor Pressure ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Amino Acid Residues ◽  
Highly Effective

AbstractFluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyper-osmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded known osmolytes used by V. parahaemolyticus to include N-N dimethylglycine (DMG) amongst others. We showed that V. parahaemolyticus requires a BCCT transporter for DMG uptake, carriers that were not known to transport DMG. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG, which was confirmed by functional complementation in E. coli strain MKH13. BccT1 was unusual in that it could uptake both compounds with methylated head groups (glycine betaine (GB), choline and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating residues for glycine betaine in BccT1. In silico modelling analysis demonstrated that glycine betaine, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four resides mutated resulted in loss of uptake of glycine betaine, DMG and ectoine. We showed three of the four residues were essential for ectoine uptake whereas only one of the residues was essential for glycine betaine uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for coordination of glycine betaine, DMG and ectoine transport.ImportanceVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high salinity conditions. In this study, we identified several novel osmolytes that are utilized by V. parahaemolyticus. We demonstrated that the compound dimethylglycine (DMG), which is abundant in the marine environment, is a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT-family carriers, which have not been shown previously to uptake this compound. BccT1 was a carrier for glycine betaine, DMG and ectoine and we identified the amino acid residues essential for coordination of these compounds. The data suggest that for BccT1, glycine betaine is more easily accommodated than ectoine in the transporter binding pocket.

49 EFFECT OF ADDING Trolox C AND ASCORBIC ACID TO RAM SPERM BEFORE CRYOPRESERVATION ON THE MOTILITY AND BINDING CAPABILITY

2016 ◽  
Vol 28 (2) ◽  
pp. 154 ◽  
Author(s):  
J. Costa ◽  
W. Lima ◽  
E. Moraes ◽  
P. Sousa ◽  
L. Ramon ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Beneficial Effects ◽  
C 30 ◽  
Motility Of Sperm ◽  
Ram Sperm ◽  
Trolox C

Different antioxidants have been tested to improve sperm quality, but distinct and consistent beneficial effects are lacking. The objective of this study was to evaluate whether the addition of different concentrations of the antioxidant Trolox C and ascorbic acid before cryopreservation could improve the binding of sperm to chicken egg perivitelline membrane (PM) after cryopreservation. Three ejaculates of ram were split and diluted with Tris egg yolk diluent to a final concentration of 200 × 106 cells mL–1, and after, the ejaculates were divided into 5 tubes. Each tube received one of the following antioxidants: control, no antioxidant; 200 μM of Trolox C; 300 μM of Trolox C; 0.05% of ascorbic acid; and 0.25% of ascorbic acid. The samples were cooled to 5°C/2 h, packaged into 0.5-mL straws, and frozen in static LN vapor for 15 min before being plunged into LN. Straws were thawed (37°C/30 s). The motility was determined using computer-assisted semen analysis. For PM binding test, PM was put in tubes with 1 mL of TALP and inseminated with 50 000 sperm. The PM and sperm were incubated for 90 min at 37°C in an atmosphere of 5% CO2 in air, and 20 min before the end of the incubation time, 10 μL of Hoechst 33342 was added in each treatment. After each PM was washed 5 times in TALP, placed under the coverslip on a slide, and evaluated by fluorescence microscopy at 400×. Spermatozoa were counted in 6 random fields of each piece of PM. Percentage data were transformed using arcsine prior analysis. Treatment differences were determine by analysis of variance and Tukey test. The total and progressive motility of sperm treated with 0.25% ascorbic acid Trolox C was higher (64.5 and 45%) than 100 μM of Trolox C (61.9 and 42.6%) and 200 μM of Trolox C (64.3 and 46.7%) and control (59.8 and 39.6%; P < 0.05), respectively. The binding test was higher when using 0.25% of ascorbic acid (155.73 cells; P < 0.05) compared with other treatments. Addition of 0.25% ascorbic acid to ram sperm before cryopreservation improved cell cryosurvival rates.

Assessment of Pomegranate Juice as Antioxidant in Extender on Cauda Epididymis Spermatozoal Quality of Buck at Refrigerated Temperature

2019 ◽  
Vol 15 (02) ◽  
pp. 26-29
Author(s):  
Amarjeet Amarjeet ◽  
C T Khasatiya ◽  
L Chaudhary
Keyword(s):  
Detrimental Effect ◽  
Egg Yolk ◽  
Antioxidant Effect ◽  
Pomegranate Juice ◽  
Antioxidant Additive ◽  
Epididymal Spermatozoa ◽  
The Dead

The present investigation was carried out to study the refrigeration preservation of the cauda epididymal retrieved spermatozoa of buck in Tris egg yolk citrate (TEYC) dilutor containing pomegranate juice as antioxidant additive. The retrieved cauda epididymal spermatozoa extended in TEYC dilutor were studied in five groups by adding different concentration of pomegranate juice as additive (0% as control T1 group and 5%, 10%, 15% and 20% as treatment T2, T3, T4 and T5 groups, respectively) and storing at refrigerated temperature up to 48 hr. The results showed that the control extender had the least dead, abnormal and HOS non-reacted sperm percent among all treatments tested and that with increasing the pomegranate juice concentration in dilutor, the percentage of the dead, abnormal and HOST non-reacted spermatozoa increased significantly. The same trend was observed at all 12 hourly storage intervals indicating its detrimental effect on epididymal sperms of bucks at refrigeration temperature. The dead, abnormal, and HOST non-reacted sperm were significantly and positively interrelated with each other (r = 0.53-0.83). It was concluded that the inclusion of pomegranate juice in TEYC dilutor did not show any beneficial/antioxidant effect on epididymal sperms of buck in fresh or refrigerated semen and in fact all the levels of pomegranate juice (5% to 20%) were detrimental to cauda epididymal spermatozoa of a buck.

scholarly journals Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

2002 ◽  
Vol 68 (11) ◽  
pp. 5647-5655 ◽  
Author(s):  
Mary Lou Mendum ◽  
Linda Tombras Smith
Keyword(s):  
Listeria Monocytogenes ◽  
Transport System ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
High Salt ◽  
Uptake System ◽  
Food Borne ◽  
Protein Mutants ◽  
Carnitine Uptake ◽  
Do So

ABSTRACT The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a Km of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a Km for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a Km for carnitine uptake of 1 to 3 μM and a V max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.

Humectant Permeability Influences Growth and Compatible Solute Uptake by Staphylococcus aureus Subjected to Osmotic Stress

2002 ◽  
Vol 65 (6) ◽  
pp. 1008-1015 ◽  
Author(s):  
ODDUR VILHELMSSON ◽  
KAREN J. MILLER
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Chloride ◽  
Water Activity ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
Foodborne Pathogen ◽  
Compatible Solute ◽  
Solute Uptake ◽  
Stressed Cells ◽  
Carnitine Uptake

The effects of different humectants (sodium chloride, sucrose, and glycerol) on the growth of and compatible solute (glycine betaine, proline, and carnitine) uptake by the osmotolerant foodborne pathogen Staphylococcus aureus were investigated. While growth in the presence of the impermeant humectants sodium chloride and sucrose induced the accumulation of proline and glycine betaine by cells, growth in the presence of the permeant humectant glycerol did not. When compatible solutes were omitted from low-water-activity media, growth was very poor in the presence of impermeant humectants. In contrast, the addition of compatible solutes had essentially no effect on growth when cells were grown in low-water-activity media containing glycerol as the humectant. Carnitine was found to accumulate to high intracellular levels in osmotically stressed cells when proline and glycine betaine were absent, making it a potentially important compatible solute for this organism.

scholarly journals Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

2000 ◽  
Vol 182 (13) ◽  
pp. 3717-3725 ◽  
Author(s):  
Eric Boncompagni ◽  
Laurence Dupont ◽  
Tam Mignot ◽  
Magne Østeräs ◽  
Annie Lambert ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Binding Protein ◽  
Compatible Solutes ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Gene Encoding ◽  
Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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