262 THE EFFECT OF TRIS AND Botu-Bov® FOR BOVINE SEXED SPERM CRYOPRESERVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 211
Author(s):  
C. P. Freitas ◽  
J. A. Dell'Aqua-Junior ◽  
F. O. Papa ◽  
M. A. Alvarenga ◽  
A. M. Crespilho ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Semen Analysis ◽  
Bos Indicus ◽  
Membrane Integrity ◽  
Reproductive Age ◽  
Freezing Process ◽  
Control Group ◽  
Sperm Cryopreservation ◽  
High Concentration

Sexing of sperm by flow cytometry has been applied worldwide. However, the sorting as well as the cryopreservation process causes damage to the sperm cells. Therefore, the sexed sperm need enhanced protection during the freezing process. Thus, the aim of the present study was to compare the effect of two different extenders for sperm cryopreservation. Two ejaculates from 10 bulls (Bos taurus and Bos indicus) of reproductive age and used in commercial AI programs were used. The ejaculates were collected with an artificial vagina and the semen was analyzed and prepared for separation by flow cytometry. After sorting, the samples were split into two groups for freezing in TRIS-egg yolk or Botu-Bov� (Biotech Botucatu, Ltda., Sao Paulo, Brazil). Sperm were evaluated by computer-aided semen analysis (CASA) and fluorescent probes to determine plasma and acrosomal membrane integrity. The fertility of the spermwas also tested in an IVF program. Statistical analysis of sperm parameters was achieved using theTukey test with a significant level of P ≤ 0.05. Embryo production data after IVF were analyzed by chi-square test. There was a significant improvement in progressive motility with the use of Botu-Bov, compared with TRIS (67.4% v. 56.7%, respectively). There was also a statistical difference for curvilinear velocity (VCL) and lateral head displacement (ALH), with TRIS showing higher values than Botu-Bov (VCL = 206.6 v. 157.3 µm s–1, and ALH = 8.0 v. 5.9 µm, respectively). According to the literature, an ALH higher than 7 µm associated with a high VCL characterizes a vigorous and disordered movement indicating hyperactivity. The sperm frozen with the Botu-Bov had a higher straight movement (STR; 85.1%) and linearity (57.4%) than the TRIS group (79.8% and 45.6%, respectively). These features indicate a straighter and more uniform movement when Botu-Bov was used. The morphological analyses using fluorescent probes showed higher proportions of intact plasma and acrosomal membranes for Botu-Bov than for TRIS (50.1% v. 39.4% and 85.3% v. 71.2% for Botu-Bov and TRIS, respectively). Blastocyst formation after IVF was 21% (68/327; blastocysts/matured oocytes) for the TRIS group. This result was statistically different from the 30% (75/252) of blastocysts obtained with Botu-Bov and from the 34% (43/128) obtained in the control group. These results suggest that Botu-Bov provides better conditions for the maintenance of the viability of frozen sexed semen than does TRIS, probably due to the high concentration of essential and nonessential amino acids present in the Botu-Bov extender. This work was supported by Sexing Technologies – Brazil and HG Lagoa da Serra.

Effect of Sorafenib on Sex Hormone Levels in Male Swiss Albino Mice

2021 ◽  
pp. 3883-3888
Author(s):  
Surekha D. Shetty ◽  
Laxminarayana Bairy K. ◽  
AM Prasad ◽  
Satheesha Nayak B. ◽  
Ashwini Aithal P.
Keyword(s):  
Body Weight ◽  
Semen Analysis ◽  
Young Patients ◽  
Differentiated Thyroid Carcinoma ◽  
Positive Control ◽  
Vital Role ◽  
Reproductive Age ◽  
Control Group ◽  
Swiss Albino Mice ◽  
Albino Mice

Background: Hormones play a vital role in initiating and maintenance of male reproductive or testicular function which includes the production of androgens and spermatozoa. Testosterone is essential for the initiation and maintenance of spermatogenesis. FSH is responsible for the stimulation of spermatogenesis. Semen analysis and hormone evaluation are essential parameters in the diagnosis of infertility in males. Objective: The aim of the present study is to evaluate the effect of sorafenib on FSH and intratesticular testosterone levels in male Swiss albino mice. Materials and Methods: The animals were segregated into control, positive control, and treatment groups (n=6). Treatment group received 25, 50 and 100 mg/kg body weight of sorafenib orally for seven consecutive days at intervals of 24 hours between two administrations. Positive control group received 100 mg/kg body weight of imatinib. The animals were sacrificed at the end of 1st, 2nd, 4th, 5th, 7th and 10th week after the last exposure to sorafenib. Results: The intratesticular testosterone level was significantly (P<0.05) reduced in treated groups and severe effect was observed on week 4th and 5th weeks. FSH level was increased significantly (P<0.05) in sorafenib treated groups of mice. Conclusion: The administration of sorafenib does affect testosterone and FSH level significantly, but this effect is reversible once the drug is withdrawn. This finding may help the clinicians to plan and address the fertility-related issues in young patients of reproductive age who are being treated with sorafenib for advanced renal cell carcinoma, hepatocellular carcinoma and differentiated thyroid carcinoma.

scholarly journals The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

2006 ◽  
Vol 31 (4) ◽  
pp. 349-356 ◽  
Author(s):  
Luiz G. Chitarra ◽  
Peter Breeuwer ◽  
Tjakko Abee ◽  
Ruud W. Bulk
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Pathogenic Bacterium ◽  
Plant Pathogenic Bacterium ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Alternatives Assessment ◽  
Heat Treated ◽  
Calcein Am

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

scholarly journals A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Atheesha Singh ◽  
Tobias George Barnard
Keyword(s):  
Flow Cytometry ◽  
Gastric Acid ◽  
Vibrio Cholerae ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Gastric Fluid ◽  
Simulated Gastric Fluid ◽  
Assessment Protocol ◽  
Pathogenic Vibrio ◽  
Vibrio Cholerae O1

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a “stressed state” due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

95 EFFECT OF CRYOPROTECTANT ADDITION AND REMOVAL AT 4°C ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL EPDIDYMAL SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 156
Author(s):  
C. Guerrero ◽  
S. Leibo ◽  
D. Paccamonti ◽  
B. Eilts ◽  
K. Bondioli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Single Step ◽  
Freezing Process ◽  
Computer Assisted ◽  
Chemical Toxicity ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa

Cryopreservation of spermatozoa harvested from the epididymides would be a means of salvaging germplasm from genetically valuable males that die unexpectedly from injury, disease, or poaching. It is well known that the addition of cryoprotective agents (CPAs) is essential for sperm survival following the freezing process. However, CPAs can cause loss in sperm viability due to osmotic damage or chemical toxicity. The objective of this study was to determine the effects of single-step addition and/or removal of glycerol (GLY) or ethylene glycol (EG) on motility and plasma membrane integrity of bovine epididymal spermatozoa. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory at 25–28°C within 4–6 h post-mortem. Epididymal spermatozoa were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and washed in Brackett-Oliphant medium by centrifugation for 5 min at 500g. Pellets were resuspended in egg yolk-Tris-glucose-citric acid monohydrate medium (EYT-GC) at a concentration of 120 × 106 cells/mL and cooled to 4°C at a rate of 0.1°C/min. Specimens were allocated to each of five treatment groups: control (no CPA), 7% GLY, and 14% GLY, 7% EG, 14% EG. Then, replicate samples were diluted 1:1 in EYT-GC medium containing twice the final desired concentration of CPA. After being exposed for 10 min, each sample was diluted directly into EYT-GC at 4°C. Motility was assessed by means of a computer assisted semen analysis system and plasma membrane integrity was determined by SYBR 14 and propidium iodide staining followed by fluorescence microscopy. Differences among treatments were analyzed using one way ANOVA (P < 0.05). The results (Table 1) show that maximum survival, as assessed by measurements of motility and membrane integrity, was achieved with spermatozoa exposed to 7% EG. Almost identical intermediate levels of survival were observed with spermatozoa exposed to 7% GLY or 14% EG. The lowest survival was observed for spermatozoa exposed to 14% GLY. The results indicate that the use of EG as a cryoprotectant may minimize toxicity and osmotic damage to fresh bovine epididymal spermatozoa. Its efficacy as a CPA is currently being determined. Table 1. Sperm motility and membrane integrity (mean ± SEM) after addition of CPA to epididymal sperm

scholarly journals Flow Cytometric Assessment of the Postantibiotic Effect of Methicillin on Staphylococcus aureus

1998 ◽  
Vol 42 (5) ◽  
pp. 1195-1199 ◽  
Author(s):  
M. T. E. Suller ◽  
D. Lloyd
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Respiratory Activity ◽  
Membrane Integrity ◽  
Postantibiotic Effect ◽  
Tetrazolium Chloride ◽  
Fluorescence Measurements ◽  
Flow Cytometric ◽  
Solid Agar

ABSTRACT The postantibiotic effect (PAE) following a 2-h exposure ofStaphylococcus aureus NCTC 6571 to methicillin (5× the MIC) was investigated with fluorescent probes, 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC), an indicator of respiratory activity, and the membrane potential-sensitive compound bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Counts of the numbers of CFU on solid agar correlated well with information gained from the CTC and DiBAC4(3) fluorescence intensity distributions obtained by flow cytometry and revealed that the postantibiotic effect was 3.1 h. Due to the capacity of flow cytometry to provide information on the heterogeneity of a bacterial population, both fluorescent probes identified the emergence of an active subpopulation 4 h after removal of the methicillin, indicating the recovery of a small percentage of the population. After removal of the methicillin and resuspension of the cells in methicillin-free medium, a further decrease in the respiratory activity and the membrane integrity of the population was observed, although the CFU counts hardly varied, indicating continued antibiotic-induced damage. Also, CTC fluorescence measurements identified numerous subpopulations during the PAE period; this suggests that the PAE is complex, with individual organisms exhibiting various degrees of recovery. Flow cytometry thus provides a rapid and sensitive alternative to traditional techniques that have been used to study PAE, with the added advantage that physiological changes can be detected as they arise.

66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN

2015 ◽  
Vol 27 (1) ◽  
pp. 126
Author(s):  
F. O. Papa ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
H. L. Resende ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Fluorescein Isothiocyanate ◽  
Treated Group ◽  
Control Group ◽  
Computer Assisted ◽  
Exact Test ◽  
Frozen Semen

The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-CryoTM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 106 of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 106 total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P > 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.

The cryoprotective effect of Ficoll on the rabbit spermatozoa quality

Zygote ◽  
2014 ◽  
Vol 23 (5) ◽  
pp. 785-794 ◽  
Author(s):  
Barbora Kuliková ◽  
Michele Di Iorio ◽  
Elena Kubovicova ◽  
Lenka Kuzelova ◽  
Nicolaia Iaffaldano ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Semen Analysis ◽  
Annexin V ◽  
Freezing Process ◽  
Control Group ◽  
Computer Assisted ◽  
Molecular Weight Polymer ◽  
Large Molecular Weight ◽  
Rabbit Spermatozoa

SummaryThe aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.

78 DIFFERENT EXTENDERS TO HARVEST EQUINE EPIDIDYMAL SPERM AND THEIR INFLUENCE ON FREEZABILITY

2013 ◽  
Vol 25 (1) ◽  
pp. 186
Author(s):  
R. F. Soares ◽  
F. O. Papa ◽  
L. C. O. Magalhães ◽  
G. A. Monteiro ◽  
I. Martin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Freezing Process ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Traumatic Injuries ◽  
Plasma Membrane Integrity ◽  
Quarter Horse ◽  
Pipette Tip

Harvesting and freezing epididymal sperm is a technology that enables the preservation of the gene pool from animals that had died either unexpectedly or because of colic conditions. This technique may also be employed in animals that have to be euthanized because of traumatic injuries. Therefore, the objective of the present study was to improve the process of freezing epididymal sperm using a freezing extender without the prior centrifugation of the samples. Twelve stallions aging between 3 and 6 years and of different breeds were used (Quarter Horse, Mangalarga, and Brazilian Jumping Horse). Stallions were castrated, and the cauda epididymides were isolated from the testis. The connective tissue was carefully dissected, and the cauda epididymides were straightened. A 200-µL pipette tip was attached to a 20-mL syringe, and the cauda epididymides were flushed using 40 mL of either (A) BotuSemen® (Nidacon, Mölndal, Sweden) or (B) BotuCrio® (Nidacon). They were then immediately processed at room temperature (25°C). Samples flushed with B were randomly subjected to either of the 2 procedures: B1) directly loaded into 0.5-mL straws or B2) centrifuged at 600g for 10 min, the supernatant was discarded, and the pellet was resuspended with B and loaded into 0.5-mL straws. The straws were kept at 5°C for 20 minutes followed by another 20 min at 6 cm above liquid nitrogen before immersion. After thawing at 46°C for 20 s the samples were analyzed by computer-assisted semen analysis (HTM – IVOS 12) and plasma membrane integrity was assessed using fluorescent probes (carboxyfluorescein diacetate and propidium iodide). Data were analyzed by ANOVA followed by the Tukey test (P < 0.05). No differences were observed for the values of total motility (A: 57.1 ± 12.35, B1: 46.3 ± 10.0, B2: 47.2 ± 12.84), progressive motility (A: 25.5 ± 9.05, B1: 21.7 ± 9.02, B2: 20.7 ± 7.20), percentage of rapids (A: 40.6 ± 15.92, B1: 30.7 ± 10.51, B2: 32.6 ± 12.39), and plasma membrane integrity (A: 47.8 ± 9.56, B1: 45.0 ± 13.81, B2: 41.3 ± 8.74). It was found that the fluid derived from epididymal secretions, which composes seminal plasma, had no influence on sperm parameters, because there was no difference among freezing protocols. Therefore, flushing equine epididymal cauda with B and freezing the samples without centrifuging can be successfully used. Both extenders (A and B) were efficient in protecting epididymal sperm throughout the freezing process.

120 Use of fixable dyes to analyze equine sperm membrane integrity and acrosome reaction after A23187 treatment

2020 ◽  
Vol 32 (2) ◽  
pp. 187
Author(s):  
I. Ortiz ◽  
M. Felix ◽  
H. Resende ◽  
C. Love ◽  
K. Hinrichs
Keyword(s):  
Flow Cytometry ◽  
Repeated Measures ◽  
Semen Analysis ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Fluorescein Isothiocyanate ◽  
Computer Assisted ◽  
Cell Agglutination ◽  
Staining Protocol ◽  
Viable Sperm

Conventional IVF is not successful in the horse, and current work is focused on factors affecting sperm capacitation in this species. Challenges arise in assessing equine sperm incubated in media containing capacitation promotors, as some of these factors cause sperm head-to-head binding (aggregation). Our preliminary microscopic findings showed that sperm aggregates are largely of viable sperm, whereas nonviable sperm individualize. Thus, data obtained using technologies that analyse only individual cells and gate out aggregates, such as flow cytometry, may not accurately represent the study population. We developed a fixable live/dead/acrosome staining protocol (LD-PSA) that minimizes sperm aggregation. The aim of this study was to evaluate the percentage of viable and acrosome-reacted equine sperm after A23187 treatment, using either LD-PSA or a standard staining protocol (PI-PSA). Sperm from 9 ejaculates were suspended in Hanks’ balanced salt solution (HBSS) and exposed for 10min at 37°C to vehicle (V) or to 10 µM A23187 (C10). The sperm were washed, resuspended in HBSS medium with added lactate and pyruvate and containing 7mgmL−1 of bovine serum albumin (BSA), and assessed immediately (0h) or incubated at 37°C for 2h (the period needed for equine sperm to respond to A23187). Motility was analysed using computer-assisted semen analysis. Each treatment was stained by PI-PSA: propidium iodide and fluorescein isothiocyanate-Pisum sativum agglutinin (PSA) in DPBS; and by LD-PSA: Live/Dead Fixable Red, paraformaldehyde 2%, Triton×1%, and FITC-PSA in Dulbecco's phosphate-buffered saline with Accumax (Stem Cell Technologies), a commercial proprietary cell agglutination inhibitor, before flow cytometric analysis. Differences were analysed using repeated-measures two-way ANOVA. The% total motile sperm (TMOT) for V and C10 treatments were 76.3±3.0 and 71.2±4.7 at 0h (P&gt;0.05), and 70.5±14.8 and 2.4±0.8 at 2h (P&lt;0.05). On flow cytometry, the percentage of events outside the gate for V sperm (0h and 2h combined) was 31.9% in PI-PSA and 21.9% in LD-PSA samples (P&lt;0.01). Measured viability in V samples was significantly lower when stained with PI-PSA than with LD-PSA at 0h (49.2±4.6 vs. 67.1±4.9) and tended to be lower (P=0.07) at 2h (44.0±4.9 vs. 55.1±2.8). Notably, the viability recorded in PI-PSA was 26 percentage points lower than was the TMOT at both 0h and 2h, indicating nonrepresentative results, as nonviable sperm should not be motile. By LD-PSA, this difference was 9 points at 0h and 15 points at 2h. Vehicle sperm showed significantly higher AR values in PI-PSA than in LD-PSA at 0h (30.4±4.0 vs. 17.7±2.4) and 2h (41.9±4.5 vs. 24.0±1.8), as did C10 sperm at 0h (28.9±2.7 vs. 18.0±2.5). The lower values for viability than total motility likely reflect agglutination of viable sperm and thus their exclusion from analysis on flow cytometry. The anti-clumping measures employed in the LD-PSA protocol were associated with increased correspondence of measured viability with TMOT. Thus, LD-PSA may offer a more accurate technique to assess viability and acrosome status of equine sperm incubated in capacitating conditions.

scholarly journals Crocin Improves the Quality of Cryopreserved Goat Semen in Different Breeds

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1101
Author(s):  
Valentina Longobardi ◽  
Gianluigi Zullo ◽  
Alessio Cotticelli ◽  
Angela Salzano ◽  
Giuseppe Albero ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Sperm Parameters ◽  
Freezing Process ◽  
Control Group ◽  
Dna Integrity ◽  
Semen Extender ◽  
Dose Trial

The effect of crocin in the semen extender before cryopreservation was evaluated on sperm parameters of 20 bucks of five different breeds: Garganica (GA), Jonica (JO), Maltese (MA), Mediterranean Red (MR) and Saanen (SA). Semen samples were centrifuged, to remove seminal plasma, divided in two aliquots and diluted with Tris-egg-yolk-based extender, containing 0 (control group) and 1 mM crocin. Crocin concentration was established after a preliminary dose trial. On fresh and frozen-thawed sperm, motility, viability, morphology, membrane integrity, DNA fragmentation and ROS levels were evaluated. The freezing process led to a decrease (p < 0.05) in all the sperm parameters recorded, confirming the deleterious effect of cryopreservation on goat semen. The most interesting result regarding the inclusion of crocin in the extender before cryopreservation was as follows: Crocin significantly improved (p < 0.05) sperm motility in all breeds, except for Mediterranean Red, compared to the control group. Furthermore, 1 mM crocin reduced percentage of spermatozoa with DNA fragmentation with a marked decrement (p < 0.05) in Garganica and Saanen, as compared to the control group. Finally, intracellular ROS decreased (p < 0.01) in the crocin-treated sperm of all breeds, as compared to the control. In conclusion, supplementation of 1 mM crocin in the extender decreased oxidative stress, improving sperm motility and the DNA integrity of frozen-thawed sperm in different breeds.

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