scholarly journals Effects of NGF Addition on Llama (Lama glama) Sperm Traits After Cooling

2021 ◽  
Vol 7 ◽  
Author(s):  
Luciana M. Sari ◽  
Renato Zampini ◽  
Francisco Gonzalez del Pino ◽  
Martin E. Argañaraz ◽  
Marcelo H. Ratto ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Active Form ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Fluorescent Marker ◽  
Seminal Plasma Proteins ◽  
Lama Glama ◽  
Components Analysis ◽  
Sperm Traits

To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that β-NGF can contribute to the improvement of sperm quality after cooling. First, β-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native β-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous β-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human β-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human β-NGF increased the percentage of total motility and vigor (p < 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous β-NGF may be profitable for the improvement of cooled llama sperm.

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.

116 EFFECT OF INCUBATION OF BOVINE EPIDIDYMAL SPERMATOZOA IN SEMINAL PLASMA PRIOR TO CRYOPRESERVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 175
Author(s):  
C. A. Guerrero ◽  
J. A. Jenkins ◽  
J. W. Lynn ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Water Bath ◽  
Sperm Chromatin ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Epididymal Sperm ◽  
Acrosome Integrity

Studies examining the influence of seminal plasma on sperm function have shown both beneficial and detrimental effects. However, its effect on pre-frozen bovine epididymal sperm (BES) has not been documented. The objective of this study was to determine the effect of a 30-min incubation of BES in bovine seminal plasma (SEMP) prior to freezing on post-thaw sperm parameters. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory (25–28°C) within 3–5 h postmortem. BES were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and split into either Treatment A, 3 mL of egg yolk Tris-based medium (EYT) (No SEMP), or Treatment B, 3 mL of SEMP, each for 30 min in a 37°C water bath. SEMP was extracted from ejaculates of mature bulls (n = 6), and then pooled and stored at −20°C until used. Sperm from both treatments were re-suspended in EYT, adjusted to 70 × 106 sperm mL−1 and cooled to 4°C at 0.1°C min−1. Samples were diluted slowly over a 30-min period with 1:1 EYT medium containing 14% glycerol and loaded into 0.5-mL straws. Straws were frozen in liquid nitrogen vapors. For sperm analyses, straws were thawed in a water bath at 37°C for 40 s. Morphology was determined by staining with eosin-nigrosin. Viability and acrosome integrity were measured simultaneously with the combination of SYBR 14, propidium iodide, and PE-PNA. Mitochondrial activity was measured by the combination of MitoTracker Red and SYBR 14. DNA integrity was determined by acridine orange using the sperm chromatin structure assay (SCSA®; SCSA Diagnostics, Inc., Brookings, SD, USA). All assays were performed by multicolor flow cytometry. Differences between treatments were analyzed using one-way ANOVA (P < 0.05). In summary, all sperm quality parameters decreased significantly after thawing in both treatments (Table 1). A significantly higher overall and progressive motility post-thaw was achieved when sperm were incubated in SEMP prior to freezing. However, no difference was detected in sperm viability, acrosome integrity, mitochondrial activity, and DNA integrity between treatments. Also, SEMP reduced the quantity of distal droplets and broken tails post-thaw over that of no SEMP. Results indicate that incubation of BES in bovine seminal plasma prior to freezing improves post-thaw sperm quality. Table 1. Effect of bovine seminal plasma (SEMP) on quality parameters (mean ± SEM) of post-thaw bovine epididymal sperm

scholarly journals From One Ejaculate to Another: Transference of Sperm Traits via Seminal Plasma Supplementation in the Ram

Biology ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 33
Author(s):  
Christine Green ◽  
Jessica P. Rickard ◽  
Simon P. de Graaf ◽  
Angela J. Crean
Keyword(s):  
Sperm Motility ◽  
Seminal Plasma ◽  
Perceived Risk ◽  
Positive Relationship ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Reproductive Technologies ◽  
Before And After ◽  
The Difference ◽  
Sperm Traits

Males can adjust sperm motility instantaneously in response to the perceived risk of sperm competition. The speed of this response suggests that sperm motility is regulated by changes in seminal plasma rather than changes in the sperm cells themselves. Hence, here we test whether inter-ejaculate variation in seminal plasma can be used to alter sperm quality prior to use in assisted reproductive technologies. We supplemented fresh ejaculates of Merino rams with seminal plasma collected from previous ‘donor’ ejaculates to test whether changes in sperm kinetics were related to the relative quality of donor to focal ejaculates. We found a positive relationship between the change in sperm traits before and after supplementation, and the difference in sperm traits between the donor and focal ejaculate. Hence, sperm motility can be either increased or decreased through the addition of seminal plasma from a superior or inferior ejaculate, respectively. This positive relationship held true even when seminal plasma was added from a previous ejaculate of the same ram, although the slope of the relationship depended on the identity of both the donor and receiver ram. These findings indicate that seminal plasma plays a key role in the control and regulation of sperm kinetics, and that sperm kinetic traits can be transferred from one ejaculate to another through seminal plasma supplementation.

Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined home-made or commercial extenders

2015 ◽  
Vol 55 (4) ◽  
pp. 551 ◽  
Author(s):  
M. Emamverdi ◽  
M. Zhandi ◽  
A. Zare Shahneh ◽  
M. Sharafi ◽  
A. Akhlaghi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Soybean Lecithin ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Flow Cytometric ◽  
Microscopic Evaluation ◽  
Open Question ◽  
Ram Sperm

The present study was designed to determine the effect of three different extenders on ram sperm quality during a freeze–thawing procedure using flow cytometric and microscopic evaluations. Several in vitro qualitative analyses of post-thawed sperm parameters including motility and velocity parameters, plasma membrane functionality, total abnormality, capacitation status, acrosome integrity, mitochondrial activity and apoptosis features were considered. In the breeding season, seven ejaculates from each Zandi ram were collected routinely twice a week. Following semen collection, samples were pooled and equally divided into three aliquots. Each aliquot was diluted and frozen with one of the following extenders: (1) Tris-based extender containing 1.5% (w/v) soybean lecithin (TSL), as a chemically defined extender, (2) Bioxcell, a commercial soybean lecithin-based extender, and (3) Tris-based extender containing 20% (v/v) egg yolk (TEY). The results of the present study indicated no differences in total [TSL (55.8 ± 2.02%) vs TEY (50.2 ± 2.02%; P < 0.05)] and progressive motility of spermatozoa [TSL (26.2 ± 1.36%) vs Bioxcell (22.4 ± 1.36%; P < 0.05)]. Semen freezing by means of TSL resulted in a higher percentage of live spermatozoa (39.42 ± 1.81%) compared with TEY (29.17 ± 1.81%; P < 0.05), and a higher percentage of functional plasma membrane (50.8 ± 192%) compared with TEY (44 ± 1.92%) and Bioxcell (38.8 ± 1.92%; P < 0.05). The effect of extenders on sperm capacitation status showed that the percentage of post-thawed capacitated spermatozoa was higher in TEY (61.9 ± 1.48%) compared with that in TSL (56.6 ± 1.48%; P < 0.05). The evaluation of post-thawed spermatozoa indicated that the percentage of live spermatozoa with active mitochondria was higher in TSL (53.05 ± 2.31%) compared with Bioxcell (45.92 ± 2.31; P < 0.05) and the percentage of intact acrosome spermatozoa was higher in TSL (84.55 ± 2.51%) compared with TEY (74.91 ± 2.51%; P < 0.05). The use of TSL and Bioxcell extenders reduced the percentage of apoptotic spermatozoa (40.82 ± 2.07% and 42.22 ± 2.07%, respectively), compared with TEY (51.34 ± 2.07%; P < 0.05). Post-thawing dead spermatozoa were increased when semen was frozen by Bioxcell (25.69 ± 1.28%). The results of this study showed that TSL extender may provide stabile milieu and conditions for ram sperm cryopreservation compared with Bioxcell and TEY extenders. Whether TSL extender can improve the artificial insemination results remains, however, an open question.

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).

Cryopreservation and egg yolk extender components modify the interaction between seminal plasma proteins and the sperm surface

2019 ◽  
Vol 140 ◽  
pp. 153-163 ◽  
Author(s):  
Rafael R.A. Ramírez-Vasquez ◽  
Adriana Cano ◽  
Federico A. Hozbor ◽  
Andreina Cesari
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Egg Yolk ◽  
Sperm Surface ◽  
Seminal Plasma Proteins

scholarly journals Spermatogenic Activity and Sperm Traits in Post-Pubertal and Adult Tomcats (Felis catus): Implication of Intra-Male Variation in Sperm Size

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 624
Author(s):  
Eliana Pintus ◽  
Martin Kadlec ◽  
Barbora Karlasová ◽  
Marek Popelka ◽  
José Luis Ros-Santaella
Keyword(s):  
Cell Function ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Sperm Head ◽  
Mitochondrial Activity ◽  
Head Width ◽  
Dna Integrity ◽  
One Year ◽  
Sperm Size ◽  
Sperm Traits

Tomcats are considered to be adults at 1 year of age, although many reach sexual maturity at an earlier age. Nevertheless, we still know little about whether the spermatogenic activity and sperm quality of mature under one-year-old tomcats differ from those of tomcats that are over one-year-old. This study aims to evaluate the spermatogenic activity, sperm traits, and their relationships in mature tomcats at two different ages. Sixteen tomcats showing complete spermatogenesis and spermatozoa in their epididymal caudae were used and classified according to their age as post-pubertal (<1 year old) or adult (˃1 year old). Our results show that adult cats have higher epididymal sperm concentration and lower coefficient of variation in sperm head width and ellipticity than post-pubertal cats. However, they do not differ in their testicular and epididymal mass, spermatogenesis, and sperm traits such as motility, mitochondrial activity, morphology, morphometry, as well as plasma membrane, acrosome, and DNA integrity. Reduced intra-male variation of sperm head ellipticity is associated with higher testis mass, epididymis mass, and sperm concentration. Interestingly, low intra-male variation in sperm head size is associated with increased Sertoli cell function and reduced post-meiotic germ cell loss. These findings increase our knowledge about feline reproductive physiology and provide new insights into the functional significance of low intra-male variation in sperm size and shape in tomcats.

scholarly journals Relationships between Seminal Plasma Metabolites, Semen Characteristics and Sperm Kinetics in Donkey (Equus asinus)

Animals ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 201
Author(s):  
Maria Antonietta Castiglione Morelli ◽  
Angela Ostuni ◽  
Brunella Giangaspero ◽  
Stefano Cecchini ◽  
Augusto Carluccio ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Relevant Information ◽  
Proton Nuclear Magnetic Resonance ◽  
Semen Parameters ◽  
Plasma Metabolites ◽  
Mitochondrial Activity ◽  
Sperm Analysis ◽  
Semen Collection ◽  
Equus Asinus

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at −80 °C for subsequent proton nuclear magnetic resonance (1H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

328 FLUORESCENT PROBE ASSESSMENT OF POST-THAW QUALITY OF RAM SPERM TREATED WITH SEMINAL PLASMA

2007 ◽  
Vol 19 (1) ◽  
pp. 279
Author(s):  
M. Rovegno ◽  
W. B. Feitosa ◽  
A. C. Brandão ◽  
A. B. Nascimento ◽  
M. A. Peres ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Fluorescent Probes ◽  
Semen Quality ◽  
Sperm Quality ◽  
Mitochondrial Activity ◽  
Published Data ◽  
Frozen Semen ◽  
Significant Difference ◽  
Negative Effect ◽  
Ram Sperm

A recently suggested alternative to improve post-thaw ovine semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryo-capacitation and helping sperm survival in the female tract. The aim of this study was to evaluate the effect of thawed ram semen incubation with seminal plasma for 15 or 30 min as assessed by the fluorescent probes, Hoechst 33342 (40 µg mL−1), propidium iodide (0.5 mg mL−1), JC-1 (76.5 µM in DMSO), and FITC-PSA (100 µg mL−1 in Dulbecco's PBS (DPBS)), for cellular viability, plasmatic damage, mitochondrial activity, and acrosomal damage, respectively. Five ejaculates were collected from 4 different animals via artificial vagina and were pooled to eliminate individual differences. After thawing, semen was divided into 2 groups, one diluted with seminal plasma (1 : 1, 30% in DPBS) and the other in DPBS (1 : 1). After 15 and 30 min, fluorescent probes were added to 25 µL of each group and 100 cells were counted under a epifluorescence microscope (Olympus IX81, motorized inverted research microscope). Spermatozoa were classified under 8 categories: alive, damaged acrosome, and high mitochondrial activity (ADH); dead, damaged acrosome, and high mitochondrial activity (DDH); alive, damaged acrosome, and poor mitochondrial activity (ADP); dead, damaged acrosome, and poor mitochondrial activity (DDP); alive, intact acrosome, and high mitochondrial activity (AIH); dead, intact acrosome, and high mitochondrial activity (DIH); alive, intact acrosome, and poor mitochondrial activity (AIP); or dead, intact acrosome, and poor mitochondrial activity (DIP). Statistical analysis was performed by Fisher test at a 5% level. The results are presented in Table 1. There was no significant difference between groups except for the AIH category which was reduced during incubation in either DPBS or SP for 15 or 30 min. These last data allow the conclusion that ram sperm is highly susceptible to cryogenic damage. Nevertheless, the high percentage of the AIH category suggests that the spermatozoa that can resist the freezing protocol remain alive and intact. Hence, we can infer that ram frozen semen has approximately half the fertilizing potential of fresh semen. In addition, we observed a negative effect of the incubation period as the decreased percentage of the AIH category was accompained by an increase of the DDP and DIP ones. We conclude that seminal plasma incubation after thawing does not benefit sperm quality. It is necessary to study the seminal plasma constitution to conclude why this experiment differed from other published data. Table 1.Spermatozoa percentages in the categories during incubation periods with or without seminal plasma This work was supported financially by FAPESP 05/55256-3

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