113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 164
Author(s):  
M. Thys ◽  
A. Van Soom ◽  
J. Dewulf ◽  
T. Rijsselaere ◽  
A. de Kruif
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Univariate Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Bovine Sperm ◽  
Sperm Characteristics ◽  
Group 2 ◽  
Group 1

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

scholarly journals Macro- and microelements in serum and seminal plasma as biomarkers for bull sperm cryotolerance

2021 ◽  
Vol 63 (1) ◽  
Author(s):  
Maja Zakošek Pipan ◽  
Petra Zrimšek ◽  
Breda Jakovac Strajn ◽  
Katarina Pavšič Vrtač ◽  
Tanja Knific ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Characteristics ◽  
Freezing And Thawing ◽  
Additional Tool ◽  
Bull Semen ◽  
Bull Sperm ◽  
Fresh Semen

ABSTRACT Background Wide variation in fertility rates is observed when using frozen bull semen, even when the bulls have met quality standards for semen production. Therefore, a simple and reliable test to assess the freezing potential of bull semen based on the analysis of fresh semen or blood would be of great value. Attention is now turning to assessment of seminal plasma components such as proteins and elements. In the present study, the concentrations of macro- and microelements in fresh bull semen plasma and in serum and their correlation with quality characteristics of fresh semen and with semen quality after freezing and thawing were determined. Ejaculates were collected from 30 mature bulls, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability and DNA fragmentation were determined on the day of collection and after freezing and thawing. The concentrations of macroelements (Na, Mg, K and Ca) and microelements (Cu, Fe, Zn and Se) were determined in the seminal plasma and serum. The semen samples were classified into satisfactory and unsatisfactory groups according to the fresh semen quality. Results Zinc and Se levels measured in serum were associated with almost all fresh and frozen-thawed semen quality characteristics, while Fe levels were associated only with acrosomal defects in fresh semen. Zinc and Fe levels in fresh seminal plasma were associated with various quality characteristics of fresh and frozen-thawed semen, while Se level in fresh seminal plasma was not associated with any of the semen quality characteristics. Conclusions Microelements were shown to be useful as biomarkers involved in the analysis of bull sperm quality and could be used as an additional tool to predict bull semen quality after freezing and thawing. Our results confirm that the analysis of Zn and Se levels in serum and Zn, Cu and Fe levels in fresh seminal plasma can provide information to discriminate between bull semen samples with spermatozoa with high or low cryotolerance.

68 DIFFERENCE IN THAWING CURVE OF STALLION FROZEN SEMEN DILUTED IN 2 EXTENDERS

2015 ◽  
Vol 27 (1) ◽  
pp. 127
Author(s):  
C. P. Freitas-Dell'aqua ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
P. M. Papa ◽  
J. A. Dell'aqua ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Freezing Process ◽  
Freezing And Thawing ◽  
Becton Dickinson ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
Group 2 ◽  
Artificial Vagina

Commercial freeze extenders have different composition and ratio of cryoprotectors; freezing and thawing protocols are different for each extender. The aim of this experiment was to observe the effect of thawing curve in stallion frozen semen with 2 commercial extenders. Two ejaculates from each of 9 stallions of different breeds (Quarter Horses and Mangalarga Marchador) were used. Semen was collected using an artificial vagina, and the ejaculate was divided into 2 groups following the manufacture's protocol: group 1 (INRA), in which the semen was diluted 1 : 1 with the extender INRA 96TM (IMV, Paillette Crista, France) and group 2 (BC), in which the semen was diluted (1 : 1) with the extender Botu-SemenTM (Botupharma, Brazil). The samples of the 2 groups were centrifuged at 600 × g for 10 min, the supernatant was discarded, and the pellet was resuspended with INRA FreezeTM (group INRA, IMV) and with BotucrioTM (group BC, Botupharma) at the concentration of sperm 100 × 106 sperm mL–1. After this, the semen was packaged in 0.5-mL straws. For each group the freezing process was carried out according to the manufacturer's instructions. The straws were thawed in a water bath with 3 different thawing curves: 37°C for 30 s (37/30), 46°C for 20 s (46/20), and 75°C for 7 s (75/7) before analysis. The aim of these rates is to keep the semen in 37°C post-thaw. The sperm kinetic analysis was performed by computerized method (CASA, HTM-IVOS, IMV, USA) and the analysis of plasma membrane integrity by flow cytometer (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA). Data of sperm kinetic and of plasma membrane integrity were compared among the 3 thawing curves for one extender using analysis of variance. Differences were considered significant at a probability level of 5%. No differences were observed in total motility (%, BC 37/30 = 72.8 ± 14.4; BC 46/20 = 70.0 ± 14.2; BC 75/7 = 70.3 ± 12.0 v. INRA 37/30 = 57.2 ± 19.1; INRA 46/20 = 50.0 ± 21.9; BC 75/7 = 58.8 ± 20.8), progressive motility (%, BC 37/30 = 36.9 ± 8.2; BC 46/20 = 34.4 ± 10.5; BC 75/7 = 33.6 ± 7.8 v. INRA 37/30 = 25.3 ± 12.7; INRA 46/20 = 21.9 ± 13.9; BC 75/7 = 28.9 ± 14.8), rapid sperm (%, BC 37/30 = 59.7 ± 16.4; BC 46/20 = 56.8 ± 17.1; BC 75/7 = 58.1 ± 14.9 v. INRA 37/30 = 38.3 ± 20.9; INRA 46/20 = 35.3 ± 22.9; BC 75/7 = 44.4 ± 23.8), and plasma membrane integrity (%, BC 37/30 = 49.1 ± 14.8; BC 46/20 = 43.1 ± 13.1; BC 75/7 = 46.7 ± 11.8 v. INRA 37/30 = 32.2 ± 10.7; INRA 46/20 = 29.6 ± 10.1; BC 75/7 = 37.4 ± 9.1) among the 3 thawing curves for INRA and BC groups. In this study, we can conclude there is no influence of the 3 tested thawing curves in sperm quality for stallion frozen semen with INRA Freeze and Botucrio extenders.

162 EFFECT OF THE SPERM SELECTION WITH ISOLATE® OR PERCOLL® ON SPERM QUALITY AND IN VITRO BOVINE EMBRYO DEVELOPMENT FOR FROZEN - THAWED SEXED AND NONSEXED SEMEN

2012 ◽  
Vol 24 (1) ◽  
pp. 193
Author(s):  
G. A. Bó ◽  
P. Rodriguez Villamil ◽  
G. Moreira ◽  
M. E. Garcia Gomez ◽  
M. Fernandez Taranco ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Sperm Selection ◽  
Production Rates ◽  
Group 4 ◽  
Sexed Semen ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

The aim of this study was to compare the effects of 2 different commercially available spermatozoa separation techniques, Isolate® (Irving-Scientific, Santa Ana, CA, USA) and Percoll® (Nutricell, São Paulo, Brazil), on sperm quality and in vitro embryo production using sexed and nonsexed semen. Oocytes (n = 5046) were obtained from slaughterhouse ovaries and fertilized with frozen-thawed sexed or nonsexed semen from the same 4 Holstein bulls. The experiment was done in 10 replicates, with all treatment groups included. Sperm quality (motility, concentration, morphology and membrane integrity) was evaluated and compared before and after sperm selection by the 2 methods. Oocytes were maturated in TCM-199 supplemented with 0.4% of BSA for 24 h in a controlled atmosphere and then selected and randomly allocated into 4 different groups. Group 1: oocytes fertilized with sexed semen selected by Percoll®; Group 2: oocytes fertilized with sexed semen selected by Isolate®; Group 3: oocytes fertilized with nonsexed semen selected by Percoll®; Group 4: oocytes fertilized with nonsexed semen selected by Isolate®. Fertilization was performed in Fert-TALP medium for 18 h under the same conditions as maturation. Presumptive zygotes were cultured for 7 days in SOF medium in a 39°C humidified incubator with 5% CO2, 5% O2 and 90% N2. Cleavage and blastocyst rates were evaluated on Day 2 and 7, respectively, after fertilization. Proportional data were transformed by square root and then analysed by ANOVA, with type of semen and sperm selection method as the main effects. Regardless of the sperm selection technique, sperm motility and percentage of normal sperm increased (P < 0.005) from the initial post-thaw parameters. For nonsexed semen, Percoll® gradient increased the recovery rate (i.e. final concentration/initial concentration × 100; 57.3 ± 2.7) compared with Isolate® (46.0 ± 1.8; P < 0.05). Furthermore, sperm selected by Isolate® presented significant improvements compared with Percoll® gradient on membrane integrity of sexed (41.0 ± 0.6 vs 38.8 ± 0.8) and nonsexed semen (60.8 ± 1.6 vs 58.8 ± 0.5; P < 0.05). Finally, blastocyst production rates were higher (P < 0.05) for sexed (Group 2: 14.0 ± 1.0) or nonsexed semen (Group 4: 22.0 ± 1.1) selected by Isolate® than for sexed (Group 1: 10.5 ± 1.5) or nonsexed semen (Group 3: 17.0 ± 2.1) selected with Percoll®. In conclusion, selection of both sexed and nonsexed semen for IVF with Isolate® resulted in higher quality sperm and higher embryo production rates than Percoll®.

scholarly journals Mitochondria Content and Activity Are Crucial Parameters for Bull Sperm Quality Evaluation

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1204
Author(s):  
Zofia E. Madeja ◽  
Marta Podralska ◽  
Agnieszka Nadel ◽  
Marcin Pszczola ◽  
Piotr Pawlak ◽  
...  
Keyword(s):  
Quality Evaluation ◽  
Semen Quality ◽  
Sperm Quality ◽  
Transcript Level ◽  
Glutathione Synthetase ◽  
Motile Sperm ◽  
Bovine Sperm ◽  
Cryopreserved Sperm ◽  
Group 2 ◽  
Group 1

Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by swim up. The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential (ΔΨm, JC-1), where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation (real-time polymerase chain reaction, PCR) of antioxidant enzymes (PRDX1, SOD1, GSS), which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 187
Author(s):  
I. Yu ◽  
Y. J. Kim ◽  
I. S. Kim ◽  
S. P. Leibo ◽  
N. Songsasen
Keyword(s):  
Pisum Sativum ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Beneficial Effects ◽  
Progressive Motility ◽  
Healthy Dogs ◽  
Sperm Survival ◽  
Acrosome Integrity ◽  
Group 1

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P &gt; 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P &lt; 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P &lt; 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.

129 IMPROVEMENT OF BOAR SPERM CRYOSURVIVAL BY THE OPTIMIZATION OF CRYOPRESERVATION CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 182
Author(s):  
J. Roca ◽  
M. Hernandez ◽  
T. Cremades ◽  
J. M. Vazquez ◽  
E. A. Martinez
Keyword(s):  
Propidium Iodide ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Limiting Factors ◽  
Commercial Application ◽  
Glycerol Concentration ◽  
Freezing And Thawing ◽  
Warming Rate ◽  
Boar Spermatozoa

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60�C min-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200�C min-1 (37�C water bath for 20 s) or ≈1800�C min-1 (70�C for 8 s). The cryopreservation condition including 2% glycerol, 40�C min-1 of cooling, and ≈1200�C min-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800�C min-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

268 IN VITRO EVALUATION OF FRESH SPERM QUALITY IN TOMCATS: A COMPARISON OF COLLECTION TECHNIQUES

2010 ◽  
Vol 22 (1) ◽  
pp. 291
Author(s):  
M. Filliers ◽  
T. Rijsselaere ◽  
P. Bossaert ◽  
P. Anastasi ◽  
M. Hoogewijs ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Sperm Head ◽  
Lower Percentage ◽  
Rank Test ◽  
Computer Assisted ◽  
Urethral Catheterization ◽  
Group 2 ◽  
Group 1

Semen can be collected from tomcats by urethral catheterization (CT) after medetomidine administration, offering a novel approach to obtain sperm for in vitro fertilization. This study was designed to determine motion characteristics of CT sperm samples by means of computer-assisted sperm analysis (CASA) and to compare sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in 17 adult cats by urethral catheterization as reported by Zambelli et al. (2008 Theriogenology 69, 485-490), after which the cat was orchidectomized. Motility (subjective and objective by means of CASA), morphology [eosin/nigrosin (E/N)], plasma membrane integrity (E/N and SYBR14-PI staining), and acrosomal status (FITC-PSA staining) of fresh CT and EP samples were evaluated and compared between both methods with a paired t-test or the Wilcoxon Rank test, dependent on normality of the data. In vitro maturation (24 h), fertilization (18 h), and culture (6h) of grade I to III oocytes were carried out as described by Pope et al. (2009 Theriogenology 71, 864-871). Twenty-four hours after in vitro insemination, fertilization rates were assessed for group 1 (CT; n = 148) and group 2 (EP; n = 159) presumptive zygotes. The distribution of presumptive zygotes between CT and EP over the different developmental stages was compared using Pearson chi-square test. Results showed that total and progressive motility as well as the percentage of normal spermatozoa were higher for EP sperm compared with CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), whereas CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other sperm parameters did not differ (P > 0.05) between collection techniques. In group 1, 84% of in vitro-matured oocytes (metaphase II) were penetrated (40.2% cleaved, 24.4% with 2 pronuclei, 12.2% with >2 pronuclei, 7.3% with expanded sperm head), whereas in group 2, penetration rate was 88.5% (42.7% cleaved, 21.8% with 2 pronuclei, 16.7% with >2 pronuclei, 7.3% with expanded sperm head). No difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the 2 methods was found. In conclusion, semen collection by means of CT yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Because CT is repeatable and easy to perform and does not require a trained male/queen in heat, it may be preferable for routine IVF experiments with fresh spermatozoa. The first author is a research fellow of the Fund for Scientific Research-Flanders (Belgium).

Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

2009 ◽  
Vol 21 (4) ◽  
pp. 571 ◽  
Author(s):  
T. Leahy ◽  
J. I. Marti ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Seminal Plasma ◽  
High Speed ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Weight Fraction ◽  
Protein Supplementation ◽  
Freezing Process ◽  
Spermatozoa Motility ◽  
Functional Integrity

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

The Effect of Diagnostic Ureterorenoscopy on Intravesical Recurrence in Patients Undergoing Nephroureterectomy for Primary Upper Tract Urinary Carcinoma

2020 ◽  
pp. 1-7
Author(s):  
Volkan İzol ◽  
Mutlu Deger ◽  
Ender Ozden ◽  
Deniz Bolat ◽  
Burak Argun ◽  
...  
Keyword(s):  
Proportional Hazards ◽  
Proportional Hazards Model ◽  
Univariate Analysis ◽  
Upper Urinary Tract ◽  
Cox Proportional Hazards ◽  
Cox Proportional Hazards Model ◽  
Recurrence Time ◽  
Tumor Biopsy ◽  
Group 2 ◽  
Group 1

<b><i>Objective:</i></b> The objective of this study is to evaluate the effect of diagnostic ureterorenoscopy (URS) prior to radical nephroureterectomy (RNU) on intravesical recurrence (IVR), in patients with primary upper urinary tract urothelial carcinoma (UTUC). <b><i>Materials and Methods:</i></b> Retrospective analysis of 354 patients, who underwent RNU for UTUC from 10 urology centers between 2005 and 2019, was performed. The primary endpoint was the occurrence of IVR after RNU. Patients were divided into URS prior to RNU (Group 1) and no URS prior to RNU (Group 2). Rates of IVR after RNU were compared, and a Cox proportional hazards model was used to evaluate potential predictors of IVR. <b><i>Results:</i></b> After exclusion, a total of 194 patients were analyzed: Group 1 <i>n</i> = 95 (49.0%) and Group 2 <i>n</i> = 99 (51.0%). In Group 1, a tumor biopsy and histopathological confirmation during URS were performed in 58 (61.1%). The mean follow-up was 39.17 ± 39.3 (range 12–250) months. In 54 (27.8%) patients, IVR was recorded after RNU, and the median recurrence time within the bladder was 10.0 (3–144) months. IVR rate was 38.9% in Group 1 versus 17.2% in Group 2 (<i>p</i> = 0.001). In Group 1, IVR rate was 43.1% in those undergoing intraoperative biopsy versus 32.4% of patients without biopsy during diagnostic URS (<i>p</i><b> =</b>0.29). Intravesical recurrence-free survival (IRFS) was longer in Group 2 compared to Group 1 (median IRFS was 111 vs. 60 months in Groups 2 and 1, respectively (<i>p</i><b></b>&#x3c; 0.001)). Univariate analysis revealed that IRFS was significantly associated with URS prior to RNU (HR: 2.9, 95% CI 1.65–5.41; <i>p</i> &#x3c; 0.001). In multivariate analysis, URS prior to RNU (HR: 3.5, 95% CI 1.74–7.16; <i>p</i> &#x3c; 0.001) was found to be an independent prognostic factor for IRFS. <b><i>Conclusion:</i></b> Diagnostic URS was associated with the poor IRFS following RNU for primary UTUC. The decision for a diagnostic URS with or without tumor biopsy should be reserved for cases where this information might influence further treatment decisions.

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