Quercetin in semen extender curtails reactive oxygen and nitrogen species and improves functional attributes of cryopreserved buck semen.

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T1 containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T2, T3, and T4 contain 10, 20, and 30 µmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 106 spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 µmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 µmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 µmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics.

Characteristics of Bachaur bull semen.

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR-National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5-5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0-5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo-osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds.

Correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation.

BACKGROUND: Dissolved oxygen (DO) in semen dilutor may lead to the production of reactive oxygen species (ROS) and buffalo sperm may become more prone to deleterious effects of ROS due to the presence of high amounts of polyunsaturated fatty acids (PUFAs) in their membranes. OBJECTIVE: To study the correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation. MATERIALS AND METHODS: Each semen sample was split into two aliquots viz., Aliquot I [diluted with Extender I (control: without deoxygenation)] and Aliquot II [diluted with Extender II: partially deoxygenated by liquid nitrogen (LN) flushing], which were diluted, filled in straws, cryopreserved and evaluated post-thaw. RESULTS: The DO levels (P < 0.05) decreased significantly after LN flushing of the extender and they increased significantly (P < 0.05) in post-thaw semen. The progressive motility, viability, hypo-osmotic swelling response, acrosomal integrity, glutathione peroxidase (GPx), total antioxidant capacity (TAC) and superoxide dismutase (SOD) decreased significantly (P < 0.05) in both control and treated semen after thawing. SOD and TAC were positively correlated in semen treated with normal extender at the pre-freeze stage; however, in semen treated with partially deoxygenated extender, no correlation was found between SOD and TAC at the pre-freeze stage. ROS and total TAC were negatively correlated in semen treated with partially deoxygenated extender at the pre-freeze stage; however, no correlation was found between ROS and TAC in control semen. CONCLUSION: The partial deoxygenation of extender affects the correlation between sperm quality parameters, antioxidants, and oxidants during different stages of semen cryopreservation. doi.org/10.54680/fr22310110712.

Exploring the role of E.coli derived enzyme, Oxyrase, as an oxygen scavenger to improve the cryotolerance of spermatozoa of Sahiwal bull.

The current study intended to optimize the concentration of Oxyrase in the semen dilutor and to evaluate its effect on freezability of spermatozoa of Sahiwal bulls. Supplementation of Oxyrase at 0.125 IU/mL concentration significantly reduced dissolved oxygen (DO) in the dilutor to 4 ppm in 16-18 min at 35 °C. For supplementation studies, a total of 24 ejaculates were categorized into poor and good ejaculates categories (n = 12 each) based on their initial progressive motility. Each ejaculate was further divided into two aliquotes. The first aliquote was diluted with tris-egg yolk extender without Oxyrase (control group) whereas, in the treatment group, Oxyrase was supplemented at the concentration of 0.125 IU/mL of extender. The parameters evaluated include cholesterol and plasma membrane phospholipids (PMP) at fresh, while IPM, acrosomal and plasma membrane integrity, cholesterol, PMP and oxidative stress parameters like lipid peroxidation (LPO), total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated at pre-freeze and post-thaw stages. The IPM and acrosomal intactness were higher (p < 0.05) in treatment group at post-thaw stage in good ejaculates. Oxyrase supplementation resulted in lower (p < 0.05) cholesterol leakage in both categories and lower (p < 0.05) LPO in good ejaculates at post-thaw stage. No statistical difference in ROS was observed between control and treatment groups at all stages whereas, level of TAC was higher (p < 0.05) in the treatment group compared to control group at post-thaw stage of both categories. Therefore, Oxyrase as an oxygen scavenging agent could preserve the post-thaw quality of Sahiwal bull spermatozoa.

Incubation with Cholesterol-loaded Cyclodextrin and Subsequent Dilution in Partially Deoxygenated Extender Improves the Freezability of Cross-bred Bull Sperm.

BACKGROUND: The cryopreservation process induces osmotic stress, membrane changes and production of reactive oxygen species resulting in damage to the spermatozoa. Together, the presence of oxygen in the extender aggravates the oxidative stress that further reduces the cryosurvival rate of sperm cells. OBJECTIVE: To study the combined effect of cholesterol loaded cyclodextrin (CLC) and partial deoxygenation on post-thaw semen quality in crossbred bulls. MATERIALS AND METHODS: A total of 18 ejaculates from three crossbred bulls with >3+ mass motility and >70% individual progressive motility were utilized for the study. Each semen sample was divided into four groups: Group I (containing extender without partial deoxygenation or CLC addition); Group II (extender containing 3 mg CLC/120X106 spermatozoa); Group III (extender containing 3 mg CLC/120X106 spermatozoa and 4 ppm dissolved oxygen (DO) level); Group IV (extender containing 3 mg CLC/120X106 spermatozoa and 6 ppm DO level). The samples in each group were finally extended to have 80×106 progressive motile sperm/mL of extender, filled and sealed in French mini straws (0.25 mL) and frozen following equilibration. The effect of CLC addition and partial deoxygenation was assessed at fresh (post-dilution), pre-freeze and post-thaw stages by evaluating various variables [sperm motility, viability, hypo-osmotic swelling (HOS) response, acrosomal integrity, capacitation status and mitochondrial membrane potential (MMP)]. RESULTS: The sperm population was significantly more positive for motility, viability, HOS response, acrosome intactness, high MMP and had less capacitation-like changes in groups supplemented with CLC and partially deoxygenation. However, the positive effect was most pronounced in the group that had extender with CLC+4 ppm DO. CONCLUSION: Partial deoxygenation of extender, and CLC addition in combination, could be part of a rationale for improving post-thaw semen quality in cross-bred bulls.

Study on correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation.

This investigation was carried out to study the correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation. Semen samples were evaluated for sperm parameters (mass motility [MM], concentration [CON], progressive motility [PM], viability [VIB], acrosomal integrity [AI] and hypo-osmotic swelling [HOS] response), antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx] and total antioxidant capacity [TAC]) and oxidants (Lipid peroxidation [LPO] and reactive oxygen species [ROS]) at fresh, pre-freeze and post-thaw stages. Sperm parameters (PM, VIB, AI and HOS response) and antioxidants (SOD, CAT and TAC) were significantly (p < .05) reduced at fresh stage, and oxidants (LPO and ROS) were significantly (p < .05) increased at pre-freeze and post-thaw stages. At fresh stage, MM was negatively correlated with LPO (p < .05), and CON was positively correlated with SOD, TAC and CAT, negatively correlated with LPO and CAT was positively (p < .01) correlated with VIB and HOS response. At pre-freeze stage, CAT was positively correlated with PM and AI (p < .05), and AI was negatively (p < .05) correlated with ROS. At post-thaw stage, CAT was positively correlated with PM, VIB, HOS response and AI,, and LPO was negatively correlated with HOS, AI and VIB. The study of correlations of these parameters at different preservation stages with bull fertility may play an important role in developing models for predicting future fertility of bulls in the absence of conception rate data.

Effect of Liquid Nitrogen Flushing of Extender on Seminal Antioxidant Profile of Murrah Buffalo during Cryopreservation.

BACKGROUND: The dissolved oxygen in the extender may act as a source for the production of reactive oxygen species that may lead to reduced seminal antioxidant profile which in turn may be responsible for impaired frozen thawed sperm quality and fertility. OBJECTIVE: To study the effect of adding liquid nitrogen into the extender on semen freezability and seminal antioxidant profile in buffalo. MATERIALS AND METHODS: Semen extender was prepared freshly and divided into two sub extenders namely, Extender I: control (non deoxygenated) and Extender II: partially deoxygenated by using LN2 flushing). The estimation of dissolved oxygen (DO) level was done in both extenders. Semen samples with mass motility of ≥ 3+ and individual progressive motility of 70% and above, collected from murrah buffalo bulls were utilized for the present study. Each semen sample was split into two group's viz., group I: diluted with extender I and group II: diluted with extender II up to 60×106 sperm/mL. The diluted semen samples were packed into French mini straws (0.25 mL), sealed with polyvinyl alcohol powder, kept for 3 h at 5°C for equilibration and then kept in automatic programmable freezer until temperature of straws reached -145°C followed by plunging into liquid nitrogen (-196°C). The evaluation of semen samples was carried out for various seminal attributes (sperm motility, live sperm count, acrosomal integrity, and hypo-osmotic swelling (HOS) response) and antioxidant profile (superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TAC)) at pre freeze and post thaw stage. RESULTS: Sperm motility, live sperm count, acrosomal integrity, HOS response were significantly (P<0.05) higher in group II as compared to group I. The average seminal SOD, GPx and TAC levels were significantly (P<0.05) higher in group II as compared to group I at pre freeze and post thaw stage. CONCLUSION: It is concluded that partial deoxygenation of the extender prior to its addition to semen enhances sperm quality in terms of sperm motility, live sperm count, acrosomal integrity, and hypo-osmotic swelling (HOS) response and also improves seminal antioxidant profile (superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TAC).

Evaluation of uterine antioxidants in bitches suffering from cystic endometrial hyperplasia-pyometra complex.

Cystic endometrial hyperplasia-pyometra complex (CEH-P) is a common disease in sexually mature bitches. Disease progression leads to oxidative stress, resulting in the depletion of uterine antioxidants and lipid peroxidation of associated cells, which further aggravates the condition. The concentration of antioxidant enzymes, the level of lipid peroxidation within the uterine tissue, and its reflection in the serum and urine need to be elucidated. The aim of this study was to analyze the concentration of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and the lipid peroxidation marker malonaldehyde (MDA) in three types of samples, i.e., serum, urine, and uterine tissue. For this purpose, 58 pyometra-affected and 44 healthy bitches were included in the present study. All animals underwent ovariohysterectomy (OVH). Our data indicated highly significant difference (p<0.01) in the antioxidant concentrations of uterine, serum and urine samples. Furthermore, there was a highly significant (p<0.01) difference in the serum levels of ferric reducing antioxidant power (FRAP) and free radical scavenging activity (FRSA) indicated poor capacity to overcome oxidative stress in the CEH-Pyometra condition. We showed that CEH-P induces oxidative stress, which further depletes the antioxidant enzyme reserves in the uterus. Thus, the weak antioxidant defence predisposes to uterine damage and disease progression. The simultaneous depletion of antioxidants and an increase in lipid peroxidation in the serum and urine may also act as early indicators of uterine pathology.

Uterine infection influences size and follicular fluid composition of the largest follicle in buffalo (Bubalus bubalis).

The effect of uterine infection on size and follicular fluid composition of the largest follicle was studied in buffalo. Reproductive tracts were collected from 102 graded Murrah buffaloes at an abattoir. Uterine infection was diagnosed by physical examination of uterine mucus, white side test and uterine cytology. Samples with pus-containing mucus, positive reaction on white side test and/or >5% neutrophils were considered to be positive for uterine infection. Diameter of the largest follicle was measured, and follicular fluid was aspirated and assayed for nitric oxide (NO), ascorbic acid (AA), cholesterol, oestradiol (E(2)) and progesterone (P(4)). Infected buffaloes had smaller-sized (p < 0.0001) largest follicles than non-infected buffaloes. Follicular fluid collected from the largest follicle in infected buffaloes had greater (p < 0.0001) NO and P(4) concentrations coincident with lesser AA (p < 0.001), cholesterol (p < 0.0001) and E(2) (p < 0.0001) concentrations. Results indicated that uterine infection has an inhibitory effect on growth of the largest follicle in buffalo. The changes in follicular fluid composition in infected buffaloes suggest that the direct effect of uterine infection on ovarian function may be mediated through an alteration in the follicular microenvironment. Greater NO and lesser AA concentrations in the follicular fluid of infected animals are novel findings.

Strategies to Minimize Various Stress-Related Freeze-Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa.

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

Reduction of dissolved oxygen in semen extender with nitrogen gassing reduces oxidative stress and improves post-thaw semen quality of bulls.

The objective of the present study was to investigate the effects of two different concentrations of dissolved oxygen (DO, 4 and 8) ppm in the extender on oxidative stress affecting plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and deoxyribonucleic acid (DNA) damage of bull spermatozoa following cryopreservation. For the experiment, nitrogen (N2) gassing of the extender for varied time intervals yielded extender with DO concentration of 4 ppm and 8 ppm (Groups II and III, respectively). For the Control (Group I) without N2 gassing, a DO concentration of 11.7 ppm was recorded. Following sample selection, ejaculates were divided into three aliquots and were extended to have 80 × 106 spermatozoa/mL of extender in the three groups. Semen samples were evaluated for reactive oxygen species (ROS), lipid peroxidation (LPO), total antioxidant capacity (TAC) and superoxide dismutase (SOD) at the fresh, pre-freeze, and post-thaw stages. Evaluation of PMI, MMP, and DNA damage were conducted on frozen-thawed samples. There were greater (P < 0.05) increase in ROS and LPO and decrease in TAC concentrations in Group I than Groups II and III. Mean values of SOD at the post-thaw stage was greater (P < 0.05) in Group II than Group I. There was a similar trend in the PMI in Groups II and III; MMP and DNA integrity in Group II was greater compared with Group I. In conclusion, results indicate there was a beneficial effect of maintaining DO concentrations at 4 rather than of 8 or 11.7 ppm in extender for sustaining post-thaw semen quality.

Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen.

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 106 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached -145 °C followed by plunging the straws into liquid nitrogen (-196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P < 0.05) in Group II as compared with the control group and Group III. Pre-freeze HOS response (%) was greater (P < 0.05) in Group II as compared with the control and Group III. At the pre-freeze stage, sperm LPO and ROS were less (P < 0.05) in Groups II and III as compared with the control and at post-thaw stage, spermatic LPO and ROS concentrations were less (P < 0.05) in Group II than in the control group and Group III. In conclusion, partial deoxygenation of extender improves sperm quality, reduces sperm LPO and ROS concentrations in buffalo during cryopreservation. Partial deoxygenation of the extender with LN2 flushing may be one of the ways for improving quality and fertility of frozen-thawed buffalo sperm.

Effect of egg yolk powder on freezability of Murrah buffalo (Bubalus bubalis) semen.

AIM: The aim of this study was to investigate the effect of commercial egg yolk powder as an alternative to fresh egg yolk on freezability of Murrah buffalo semen. MATERIALS AND METHODS: Semen samples (12) from 3 Murrah buffaloes (4 from each bull) with mass motility (≥3+) and total motility (70% and above) were utilized in this study. Immediately after collection, each sample was divided into four groups. Groups I was diluted up to 60×10(6) sperm/ml with tris extender containing 10% fresh egg yolk and Groups II, III, and IV were diluted up to 60×10(6) sperm/ml with tris extender containing 2%, 4%, and 6% egg yolk powder, respectively. Semen samples were processed and cryopreserved followed by examination of frozen semen samples after 24 h. Semen samples from each group were evaluated for total motility, viability, acrosomal integrity, abnormality, and hypo-osmotic swelling test (HOST) response after dilution, pre-freeze, and post-thaw stage. RESULTS: Pre-freeze total motility was significantly (p<0.05) higher in Groups III and IV as compared to Groups I and II, and post-thaw total motility was significantly (p<0.01) higher in Group III as compared to other three groups. Viability was significantly (p<0.05) higher in Groups II, III, and IV than Group I at the pre-freeze stage. Significantly (p<0.01) higher viability and acrosomal integrity were recorded in Group III as compared to other three groups at the post-thaw stage. Abnormality was significantly (p<0.05) higher in Group IV than other three groups. HOST response was significantly (p<0.05) higher in Groups II and III than Groups I and IV at the pre-freeze and post-thaw stages. CONCLUSION: Addition of egg yolk powder at 4% level yielded significantly better results in terms of post-thaw semen quality as compared to the fresh egg yolk and other concentrations of egg yolk powder (2% and 6%).

Effect of incubation on freezability of cholesterol-loaded cyclodextrin treated buffalo (Bubalus bubalis) spermatozoa.

AIM: The aim of this study was to investigate the effect of incubation on freezability of cholesterol loaded cyclodextrin (CLC) treated buffalo spermatozoa. MATERIALS AND METHODS: Semen samples with mass motility of 3+ and greater, collected from Murrah buffalo bulls were utilized. Immediately after collection, four equal groups of semen sample were made. Group I was kept as control and diluted with Tris upto concentration of 60×10(6) sperm/ml, where as Groups II, III, and IV were treated with CLC at 3 mg/120× 10(6) spermatozoa, incubated at 37°C for action of CLC for 10, 15 and 20 min, respectively, and diluted with tris upto concentration of 60×10(6) sperm/ml. All groups were subjected to equilibration and freezing. The evaluation of semen samples from all groups was carried out at fresh, pre-freeze and post-thaw stage for progressive motility, viability and hypo-osmotic swelling response (HOS response). RESULTS: At the pre-freeze stage, significantly (p<0.05) higher percentage of progressive motility and viability was observed in treatment groups as compared to control with no significant difference among treatment groups. HOS response was significantly (p<0.05) higher in treatment groups as compared to control at pre-freeze stage. At post-thaw stage, significantly (p<0.05) higher percentage of progressive motility, viability and HOS response was recorded in Group II as compared to control and other treatment groups (III and IV). Group II retained significant post-thaw motility and viability at various post-thaw incubation periods. CONCLUSION: Incubation period of 10 min for CLC treated buffalo spermatozoa yielded significantly higher results in terms of freezability as compared to incubation for 15 and 20 min.

Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa.

Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen-thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen-thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×10(6)) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes.

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/ synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.

A ten-year review of Candida sepsis and mortality in burn patients.

A retrospective analysis of Candida sepsis was carried out in 1722 burn patients admitted to this center from 1975 to 1984. Cultures were positive for Candida in 233 (13.5%) of these patients during their hospitalization. Candidemia was present in 70 (4.0%) of the 1722 patients. Of the 70 patients with candidemia, 38 (54%) died. However, only 11 patients (15.7%) died of Candida sepsis or mixed Candida and bacterial sepsis (less than 1% of the total patient population). The remaining 27 patients who had candidemia died of bacterial septicemia or organ system failure. The low incidence of Candida and the low incidence of mortality due to Candida was attributed to a comprehensive program of prevention, detection, and treatment. Early initiation of treatment with amphotericin B was an important aspect of the program.

Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling.

BACKGROUND: Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). STUDY DESIGN: We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. RESULTS: We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. CONCLUSIONS: The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.

Lymphoedema of lower extremities following depridement of extensive full skin thickness burns.

Burns involving skin and subcutaneous tissue are rarely associated with lymphoedema of the extremity due to the intact deep lymphatic system. We report an example of lymphoedema of the lower extremity in a burn patient 6 months after skin grafting.

Burns of the lymphoedematous extremity.

In most patients with lymphoedema, neither a medical nor a surgical approach can provide a cure. Due to the progressive nature of the disease, it is imperative that every attempt should be made at controlling oedema and preventing infection. As burn injury to the lymphoedematous extremity is associated with significant morbidity, we report a patient with deep burns affecting a lymphoedematous extremity.