scholarly journals The in vitro effect of kanamycin on the behaviour of bovine spermatozoa

2019 ◽  
Vol 1 (4) ◽  
pp. 36-40
Author(s):  
Michal Ďuračka
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
In Vitro Cultivation ◽  
Sperm Analysis ◽  
Time Dependent Effect ◽  
Bovine Spermatozoa ◽  
And Function ◽  
Vitro Effect

The use of antibiotics is a common part of animal biotechnologies. Especially, the use of antibiotics in semen extenders is necessary. However, the effect of antibiotics on the spermatozoa structure and function is still not completely examined. Therefore, the aim of our study was to investigate the effect of kanamycin on bovine spermatozoa at concentrations of 80 and 160 µg/mL during the 24 h in vitro cultivation. Semen samples were collected from clinically healthy Holstein-Friesian bulls. At times of 0, 2 and 24 h the motility assessment, mitochondrial activity, acrosomal and membrane integrity evaluation were performed. The sperm motility was measured using the Computer-assisted sperm analysis (CASA). Mitochondrial activity was evaluated through the Mitochondrial Toxicity Test (MTT). The acrosomal status was determined using the fast green/rose bengal staining on slides. Similarly, the membrane integrity was analysed using the eosin-nigrosin staining. Our results revealed the dose- and time-dependent effect of kanamycin under the in vitro conditions. In conclusion, the selected concentrations of kanamycin may have adverse effects on the motility, mitochondrial activity, acrosomal and membrane integrity during semen processing. Considering the relatively low concentrations used, we do not recommend to use kanamycin as a supplement in bovine semen extenders.

scholarly journals Effectiveness of tes-tris or tris association with low density lipoprotein on in vitro longevity of refrigerated buffalo semen

2020 ◽  
Vol 72 (3) ◽  
pp. 729-736
Author(s):  
J. Almeida ◽  
M.F. Brito ◽  
V.A.B. Becerra ◽  
B.P. Neves ◽  
P.A. Auler ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Membrane Integrity ◽  
Density Lipoprotein ◽  
Fixed Time ◽  
Computer Assisted ◽  
Low Density ◽  
Promising Alternative ◽  
Sperm Analysis ◽  
Buffalo Semen

ABSTRACT This study investigated in vitro the efficacy of four different extenders (TES-TRIS and TRIS with LDL low-density lipoprotein at concentrations of 10 or 5%) on the longevity of buffalo sperm in the refrigeration process at 5ºC. Sperm motility was assessed every 24 hours up to 72 hours of incubation using computer assisted sperm analysis and sperm membrane integrity was examined by the hypoosmotic test (HOST) at T1, T24, T48 and T72 hours. Eleven buffaloes (1 ejaculate per buffalo) of the Murrah breed were used, ranging in age from 4 to 5 years. Immediately after collection, each ejaculate was fractionated into 4 aliquots, and each aliquot was diluted in one of four diluents to obtain 50x106SPTZ/mL. The samples were packed in 0.5mL straws and refrigerated (-0.25°C/min) to 5°C and maintained at this temperature until evaluation. Prior to evaluation the samples were heated at 37°C for 30 seconds. The statistical package used for analysis was STATA 12.0 "Statistical Analysis Software" and means were compared by the Friedman test (P<0.05). The results of sperm kinetics and HOST indicate that the TRIS diluent with 10% LDL could be a promising alternative for semen refrigeration at 5ºC, to be used in conventional and fixed time artificial insemination.

240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS

2011 ◽  
Vol 23 (1) ◽  
pp. 218
Author(s):  
E. G. A. Perez ◽  
M. Nichi ◽  
C. A. Baptista Sobrinho ◽  
P. A. A. Góes ◽  
A. Dalmazzo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Structural Damage ◽  
Membrane Integrity ◽  
Domestic Cat ◽  
Lower Percentage ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Suitable Model ◽  
Sperm Analysis

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

scholarly journals Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation

2021 ◽  
Vol 9 ◽  
Author(s):  
María Milagros Giaccagli ◽  
Matías Daniel Gómez-Elías ◽  
Jael Dafne Herzfeld ◽  
Clara Isabel Marín-Briggiler ◽  
Patricia Sara Cuasnicú ◽  
...  
Keyword(s):  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Specific Staining ◽  
Sperm Analysis ◽  
Functional Changes ◽  
Fertilizing Ability ◽  
Sperm Fertilizing Ability

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

12 COMPUTER-ASSISTED SPERM ANALYSIS OF FRESH EPIDIDYMAL CAT SPERMATOZOA AND THE IMPACT OF COOLED STORAGE (4°C) ON SPERM QUALITY

2008 ◽  
Vol 20 (1) ◽  
pp. 86
Author(s):  
M. Filliers ◽  
T. Rijsselaere ◽  
P. Bossaert ◽  
V. De Causmaecker ◽  
J. Dewulf ◽  
...  
Keyword(s):  
Reference Values ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Sperm Analysis ◽  
Computer Assisted Sperm Analysis ◽  
The Impact ◽  
Motility Parameters

Feline epididymal sperm is commonly used for in vitro fertilization. It also yields the opportunity to conserve genetic material from valuable males that suddenly die. Epididymal sperm quality parameters vary considerably among laboratories, implicating the need for objective evaluation methods. The aim of the present study was to describe reference values of computer-assisted sperm analysis (CASA) parameters of fresh epididymal cat sperm and to assess the effect of prolonged cooled storage (4�C) on various sample characteristics. Epididymides obtained from tomcats after routine orchiectomy (2–4 pairs/replicate) were sliced to release spermatozoa. The sperm suspension was placed on a 2-layer gradient and, after centrifugation, the sperm pellet was recovered. In Experiment 1 (20 replicates), sperm motility parameters were assessed immediately after retrieval (T0) using the Hamilton Thorne analyzer Ceros 12.1 (HTR; Hamilton Thorne Biosciences, Beverly, MA, USA). In Experiment 2, fresh (T0) sperm samples (4 replicates) were evaluated for motility parameters (HTR), acrosomal status (FITC-Pisum sativum agglutinin staining), morphology (eosin/nigrosin (E/N) staining), and membrane integrity (E/N and SYBR�-14-propidium iodide staining; Molecular Probes, Inc., Eugene, OR, USA). After addition (1:2) of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled and reassessed on Days 1 (T1), 3 (T3), 5 (T5), 7 (T7), and 10 (T10). Results were analyzed in a mixed linear model, with replicate as random factor and time as fixed effect (S-PLUS 7.0; Insightful Corp., Seattle, WA, USA). Results of Experiment 1 were as follows (mean � SD): motility (MOT): 80.8% � 23.5; progressive motility (PMOT): 69.9% � 23.2; velocity average pathway (VAP): 98.7 µm s–1 � 24.2; velocity straight line (VSL): 89.3 µm s–1 � 25.4; velocity curved line (VCL): 134.8 µm s–1 � 31.9; amplitude lateral head (ALH): 4.3 µm � 2.0; beat cross frequency (BCF): 34.6 Hz � 7.0; and straightness (STR): 89.6% � 6.6. In Experiment 2, MOT, PMOT, VAP, VSL, VCL, BCF, and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) compared to fresh samples, starting from T1, T3, T5, T7, T5, T3, and T1, respectively. In contrast, STR, ALH, membrane integrity, and the percentage of acrosome-intact spermatozoa were not affected (P > 0.05) by cooled storage. To summarize, we have presented a set of reference values for CASA-parameters of fresh, epididymal cat spermatozoa. Cooled storage impaired most motility parameters and lowered the percentage of normal spermatozoa, but did not influence membrane integrity or acrosomal status. The effect of cooled storage on DNA fragmentation of sperm and its subsequent influence on in vitro embryo development require further investigation.

88 SELECTION OF A CRYOSURVIVAL SPERM POPULATION DOES NOT IMPROVE THE IN VITRO PENETRATING ABILITY OF FROZEN - THAWED BOAR SPERMATOZOA

2008 ◽  
Vol 20 (1) ◽  
pp. 124
Author(s):  
M. L. Perals ◽  
M. A. Gil ◽  
E. M. Garcia ◽  
J. Sanchez-Osorio ◽  
J. M. Vázquez ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Molecular Probes ◽  
Computer Assisted ◽  
Immature Oocytes ◽  
Sperm Analysis ◽  
Boar Spermatozoa ◽  
Casa System ◽  
Selection Of

Boars can be classified as good or bad sperm freezers according to their sperm cryosurvival. Different sperm selection techniques, such as PureSperm� (PS; MidAtlantic Diagnostics, Inc., Mount Laurel, NJ, USA), have been developed to improve functional competence of spermatozoa. The aim of this experimental study was to assess the ability of PS for improving the in vitro penetrating ability of frozen–thawed boar spermatozoa from good and bad sperm freezers. The sperm-rich fractions from two boars, good (Boar A) and bad (Boar B) freezers, were extended in a lactose/eggyolk/ glycerol/Equex Stem (Noba Chemical Sales, Inc., Scituate, ME, USA) mixture (1 � 109 sperm mL–1), dispensed into 0.5-mL straws, and frozen using a programmable cell freezer. After thawing (1.200�C min–1), semen from each boar was split into two aliquots of 500 µL. One aliquot was used as the control. The second was placed into a tube of PS gradient (90%/45%) and centrifuged at 425g for 20 min; the pellet re-suspended in 1 mL of BTS and re-centrifuged at 320g for 10 min (PS sample). Control and PS samples were diluted in supplemented TCM-199 (TCMm; Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485) at 200 � 106 sperm mL–1. Sperm survival (SV) was assessed afterTCMm dilution according to progressive sperm motility (PSM, %) using a computer-assisted sperm analysis (CASA) system (ISAS�), and plasma and acrosome membrane integrity (PMI; %) by flow cytometry (SYBR�-14/PE-PNA/PI; Molecular Probes, Leiden, The Netherlands). A homologous in vitro penetration (hIVP) assay, using immature oocytes (20 oocytes/2 mL TCMm supplemented with caffeine and calcium lactate), was used to assess sperm penetrating ability (Martinez et al. 1993 Theriogenology 40, 547–557). A total of 960 immature oocytes were inseminated (200 � 103 sperm/oocyte) in 3 batches. After 18 h of co-incubation at 39�C under 5% CO2 in air, the oocytes were washed, mounted on slides, fixed with ethanol:acetic acid (3:1, v/v) for 48 h, stained with 1% lacmoid, and examined under a phase contrast microscope (�400). Oocytes with swollen or unswollen heads of sperm found in the vitellus were considered as penetrated. Sperm penetrability ability (SPA) was assessed according to penetration rate (PR) and the mean number of sperm per oocyte (S/O). Data were analyzed using a PROMIXED model and expressed as mean � SEM. Boar A showed better (P ≤ 0.01) results for both SV and SPA parameters than boar B, independent of sperm treatment. PureSperm improved (P ≤ 0.05) PSM and PMI in both boar A (control v. PS: 48.0 � 5.8 v. 66.5 � 3.6 and 63.1 � 7.7 v. 88.4 � 1.3, respectively) and boar B (12.3 � 1.2 v. 22.2 � 3.7 and 44.3 � 3.5 v. 58.7 � 7.0, respectively). However, no differences (P ≥ 0.05) were observed in PR and S/O in either boar A (71.2 � 3.4 v. 78.3 � 3.1 and 5.0 � 0.4 v. 5.2 � 0.4, respectively) or boar B (34.3 � 3.6 v. 37.3 � 3.9 and 1.5 � 0.1 v. 1.5 � 0.1, respectively). In conclusion, under our laboratory conditions, PureSperm selection improves sperm quality but not in vitro penetrating ability of frozen–thawed spermatozoa of both good and bad sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

Insights into soy lecithin and egg yolk-based extenders for chilling canine spermatozoa

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Andressa Dalmazzo ◽  
Daniel de Souza Ramos Angrimani ◽  
João Diego A. Losano ◽  
Carolina C. Rocha ◽  
Carlos A. B. Sobrinho ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Compensatory Mechanism ◽  
Soy Lecithin ◽  
Sperm Analysis ◽  
Tbars Assay

SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

scholarly journals The Effect of Penicillin on the Vitality of Bull Spermatozoa

2020 ◽  
Vol 2 (3) ◽  
pp. 30-34
Author(s):  
Ján Kováč ◽  
Nishonoy Khasanova ◽  
Tomáš Slanina ◽  
Peter Massányi ◽  
Eva Tvrdá
Keyword(s):  
In Vitro Culture ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Lower Amount ◽  
Mitochondrial Activity ◽  
Sperm Analysis ◽  
Sperm Motion ◽  
Acrosomal Integrity

Antibiotic supplementation into semen extenders is an important way to control several microorganisms that can affect semen quality by their presence. The objective of the present work is to estimate the effect of two different concentrations (300 µg/mL and 600 µg/mL) of penicillin on the selected quality parameters of spermatozoa collected from bulls (motility, mitochondrial activity, acrosome integrity and membrane integrity) after 0, 2 and 24 h of in vitro culture. Sperm motion was examined using HTM IVOS computer-aided sperm analysis (CASA), cell viability was assessed with the metabolic activity (MTT) assay. The acrosomal integrity was evaluated following the fast green – rose bengal staining protocol and the eosin – nigrosin staining method was used to assess the functional integrity of the sperm membrane. Our results indicate that penicillin at lower amount significantly (p>0.05) decreased the sperm motility, mitochondrial activity and membrane integrity after 24 h of in vitro culture. Supplementation of higher doses of this substance led to a significant decrease of the sperm motion during 0, 2 (p>0.05) as well as after 24 h (p>0.01), of the viability after 2 h (p>0.05) and 24 h (p>0.01), of the acrosomal integrity after 24 h (p>0.05) and of the membrane integrity at 24 h (p>0.001) too. We can consider, that the effect of penicillin addition to bovine spermatozoa during in vitro incubation is time and dose dependent.  

scholarly journals In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257766
Author(s):  
Zuzana Knazicka ◽  
Hana Duranova ◽  
Veronika Fialkova ◽  
Michal Miskeje ◽  
Tomas Jambor ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ferrous Sulphate ◽  
Annexin V ◽  
Computer Assisted ◽  
Spermatozoa Motility ◽  
Bovine Spermatozoa ◽  
High Concentrations ◽  
Vitro Effect ◽  
Motility Parameters

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 μM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 μM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

91 TREATMENT OF GOAT SPERM WITH CATALASE TO IMPROVE POST-THAW QUALITY

2011 ◽  
Vol 23 (1) ◽  
pp. 151
Author(s):  
R. O. C. Silva ◽  
M. Nichi ◽  
E. G. A. Perez ◽  
P. A. A. Góes ◽  
A. Dalmazzo ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Structural Damage ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Thiobarbituric Acid ◽  
Lower Percentage ◽  
Sperm Chromatin ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Sperm Analysis

Semen cryopreservation is extremely important to the use of reproductive biotechnologies in goats. However, studies indicate that cryopreservation may lead to increased oxidative stress which may cause structural damage to biomolecules, DNA, lipids, carbohydrates, and proteins, as well as other cellular components. Previous studies performed by our group indicate fresh goat sperm is highly susceptible to the attack of hydrogen peroxide. Therefore, the treatment with hydrogen peroxide scavengers would be an alternative to improve post-thaw sperm quality in goats. The aim of the present study was to evaluate the efficiency of catalase, an important hydrogen peroxide scavenger, to improve post-thaw quality in cryopreserved goat semen samples. Semen samples from 5 adult goats were collected and cryopreserved (Botu-Bov®, Biotech Ltda.). After thawing, samples were incubated (2 h, 37°C) with 0, 60, 120, and 240 UI mL–1 of catalase. Samples were then analysed for motility using the computer assisted sperm analysis (CASA); the 3–3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (Fast green/Bengal rose), as an index of acrosome integrity; sperm chromatin structure assay, as an index of DNA fragmentation; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that catalase treatment after thawing played a role on improving mitochondrial activity. Samples treated with 240 UI mL–1 showed lower percentage of sperm showing low mitochondrial activity when compared with samples treated with 0 and 120 UI mL–1 of catalase (6.5 ± 2.3, 17.2 ± 3.5, and 10.0 ± 1.3%, respectively). However, no effect of catalase was observed on any of the other variables studied. Results indicate that catalase, despite its beneficial effect on mitochondrial activity, does not influence positively on sperm quality after thawing. A hypothesis to explain such results would be that because of seminal plasma dilution with the extender, the antioxidants were also diluted. Therefore, the antioxidant protection would be impaired and the most deleterious reactive oxygen species, as observed in fresh semen, would also be different depending on the semen extender used because sperm are extremely dependent on the extracellular environment due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in the membrane. A study performed by our group confirms such a hypothesis. Possibly, the treatment with catalase would be more effective if performed before cryopreservation. Also, it is possible that the use of different antioxidants would provide better results. Thanks to Nutricell for the media used and CAPES for financial support.

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