scholarly journals Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1065
Author(s):  
Valentina Longobardi ◽  
Michal A. Kosior ◽  
Nunzia Pagano ◽  
Gerardo Fatone ◽  
Alessia Staropoli ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Glycine Betaine ◽  
Low Fertility ◽  
High Fertility ◽  
Over Expression ◽  
Bull Semen ◽  
Before And After ◽  
Associated Proteins ◽  
Cellular Compartments

Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions

2022 ◽  
Author(s):  
Róisín Ann Griffin ◽  
Aleona Swegen ◽  
Mark A Baker ◽  
Rachel Ann Ogle ◽  
Nathan Smith ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Low Fertility ◽  
High Fertility ◽  
Short Term ◽  
Sperm Binding ◽  
Merocyanine 540 ◽  
Seminal Plasma Proteins ◽  
Assessment Strategies

Abstract Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days post-breeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations, and to determine the biological mechanisms governing such differences. Using LC–MS/MS, we compared the proteomic profile of semen samples collected from commercially “fertile” stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (p ≤ 0.05). Assessment of intra- and inter-stallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein), were significantly more abundant during “high-fertility” periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1] and clusterin), were significantly more abundant during “low-fertility” periods. We hypothesised that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (p ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

148 Bull spermatozoa uptake of extracellular vesicles from bovine seminal plasma

2020 ◽  
Vol 32 (2) ◽  
pp. 200 ◽  
Author(s):  
N. Pagano ◽  
M. A. Kosior ◽  
B. Gasparrini ◽  
V. Longobardi ◽  
C. De Canditiis ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Extracellular Vesicles ◽  
Seminal Plasma ◽  
Noncoding Rna ◽  
Low Fertility ◽  
Fluorescence Signal ◽  
High Fertility ◽  
Spermatozoa Motility ◽  
Rna Molecules ◽  
Bull Semen

Extracellular vesicles (EV) are important mediators of intercellular communication because they transfer microRNA (miRNA) that are able to repress translation of mRNA. Their presence in seminal plasma suggests a role in sperm fertility. It is known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility (Killian et al. 1993 Biol. Reprod. 49, 1202-1207). In addition, a difference in miRNA content between high and low spermatozoa motility has been observed in bulls, highlighting a potential role of EV on fertility (Capra et al. 2017 BMC Genom. 18, 14). We hypothesised that co-incubation of sperm of low-fertility bulls with EV isolated from the seminal plasma of high-fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary study was carried out to investigate the presence and type of EV in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls collected weekly by artificial vagina were centrifuged at 1600×g for 10min to pellet spermatozoa and then centrifuged again at 2400×g for 30min to eliminate cell debris and large vesicles. After centrifugation, supernatants were collected and filtered twice (0.45 and 0.22µm) and stored at −80°C. A double ultracentrifugation at 100 000×g for 1h was performed, and pellets resuspended in a small volume of Tris buffer were kept at −80°C until used. Three ejaculates of the same bull were pooled to detect the concentration and size of EV by Nanosight Instruments. To trace the interaction with spermatozoa by fluorescence microscopy, EV were labeled with PKH26 dye and a dose-response curve in three replicates was performed. A suspension of 1×106 spermmL−1 was co-incubated with 200 or 400×106 EV labelled with pKH26 for 30, 60, 90, 120, 150, and 180min at 38.5°C. The end point of incubation was at 24h. Internalisation of EV was assessed using confocal microscopy. Our results showed that the size of EV ranged from 145.1 to 187.7nm, with an average of 166±29nm. For all seminal plasma samples, the number of EV ranged from 3.62 to 6.08×1013 particlesmL−1, with an average of 4.37±0.61×1013. Based on size, these EV can be categorised as shedding vesicles. Confocal microscopy was set to take fluorescent images at different planes scanned every 0.12µm from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after co-incubation with 200×106 EV. At the concentration of 400×106 EV, up to 60min no signal was detectable, whereas at 90min spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120min, the signal was within the acrosome, and at 180min the spermatozoa were stained for the whole length, supposing a distribution of incorporated EV throughout all the cell. At 24h, the fluorescence signal decreased. In conclusion, this is the first study to demonstrate that bull spermatozoa incorporate EV from bull semen. We hypothesise that a transfer of molecules, such as miRNA and other noncoding RNA molecules, from EV to spermatozoa is probably involved in sperm fertility.

scholarly journals Spermatozoa Obtained From Alpaca vas deferens. Effects of Seminal Plasma Added at Post-thawing

2021 ◽  
Vol 8 ◽  
Author(s):  
Eduardo G. Aisen ◽  
Wilfredo Huanca López ◽  
Manuel G. Pérez Durand ◽  
Edita Torres Mamani ◽  
Juan C. Villanueva Mori ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Artificial Insemination ◽  
Reproductive Tract ◽  
Vas Deferens ◽  
Female Reproductive Tract ◽  
Low Fertility ◽  
Sperm Cells ◽  
South American Camelids ◽  
Thawing Process

The viscous seminal plasma (SP) is currently a major impediment to the handling of ejaculate and the development of some biotechnologies in South American camelids. The vas deferens-collected spermatozoa of alpacas is a useful technique to avoid this problem. On the other hand, SP contains a large protein component that has been implicated in the function of spermatozoa within the female reproductive tract. In this sense, the low fertility achieved using transcervical insemination with frozen-thawed spermatozoa in alpacas could be improved by adding SP. This study aimed to evaluate the effect of the whole SP on some in vitro parameters of alpaca spermatozoa after the freezing-thawing-process and the fertility after artificial insemination. It would contribute to a better understanding of the interaction between thawed sperm cells and SP. Spermatozoa were obtained by surgically diverted vas deferens. The samples were diluted with a Tris-based extender, packaged in straws, and frozen. At thawing, each straw was divided into two post-thawing conditions: with the addition of 10% of PBS (control) or with 10% SP (treatment). The sperm cells were evaluated using dynamic parameters, sperm cell morphology, and morphometry. Fertility was assessed by an artificial insemination trial. All in vitro parameters were analyzed by ANOVA. A heterogeneity test was scheduled for the fertility trial. After the freezing-thawing process, motility and plasma membrane functionality was improved when SP was added. No differences were found for post-thaw viability between the control and treatment samples. The percentage of normal cells was higher with SP at post-thawing, and a decrease of the presence of bent tailed spermatozoa with a droplet in the SP group was observed. The length of the head spermatozoa was 3.4% higher in the samples with PBS compared to those in which SP was added. Females pregnant at day 25 post-insemination were 0/12 (with SP inside the straw) and 1/10 (without SP inside the straw). In conclusion, the presence of 10% SP at post-thawing improves sperm cells' motility, functionality, and morphology, indicating that it would be beneficial to improve the frozen-thawed alpaca's physiology spermatozoa. More fertility trials must be developed to increase this knowledge.

scholarly journals Malondialdehyde Level in Seminal Plasma of Cryopreserved Holstein Bull Semen after Addition of Zinc, Cysteine, Prostaglandin F2α and their Combination in vitro

2020 ◽  
Vol 13 (1) ◽  
pp. 97-100
Author(s):  
M. R. G. Al-Dahan ◽  
A. F. Majeed ◽  
M. A. Abed ◽  
F. Ibrahim ◽  
K. J. yahya
Keyword(s):  
Seminal Plasma ◽  
Zinc Sulphate ◽  
Prostaglandin F2α ◽  
Treated Group ◽  
Control Group ◽  
Bull Semen ◽  
Holstein Bull ◽  
Holstein Bulls ◽  
Combination Treated Group

The study was conducted to know the level of Malondialdehyde (MDA) in seminal plasma of cryopreserved semen of Holstein bulls after addition of zinc sulphate, cysteine, PGF2α and their combination in vitro. Semen was collected from 7 Holstein bulls, presented in Artificial insemination Center which belonged to the Directorate of Animal Resources/ Ministry of Agriculture at Abu-Graib at the west of Baghdad. Pooled semen were diluted with Tris- based extender and divided into five parts. The first part (T1) serve as a control (without addition). The 2nd part (T2) added to it zinc sulphate (0,576 mmol/ ml). The 3rd part (T3) added to it cysteine (5 mmol/ ml). The 4th part (T4) added to it PGF2α (37.5 pg/ ml). while the 5th part added to it a combination of previous substances at the same concentration. They packed in straws and cryopreserved in a liquid nitrogen and after 30, 60 and 90 days. Seminal plasma when isolated to measure the level of MDA. The results showed a significant decrease (P>0.01) in MDA level in the combination treated group (zinc, cysteine and PGF2α) 0.450 ± 0.11 (mmol/ ml) as compared with control group 1.025 ± 0.38 (mmol/ ml), zinc 0.867 ± 0.12 (mmol/ ml), cysteine 1.06 ± 0.12 (mmol/ ml) and PGF2α group 0.968 ± 0.17 (mmol/ ml) respectively. It was concluded from this study that addition of a combination of zinc, cysteine and PGF2α to the Holstein bull semen could decrease the level of MDA which might be due to the synergistic effect of these substances.

92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE

2006 ◽  
Vol 18 (2) ◽  
pp. 154
Author(s):  
J. Gadea ◽  
S. Martínez-Miró ◽  
G. Decuadro-Hansen ◽  
C. Matás
Keyword(s):  
Seminal Plasma ◽  
Acrosome Reaction ◽  
Sperm Viability ◽  
Lipid Packing ◽  
Lipid Disorder ◽  
Merocyanine 540 ◽  
Centrifuge Tube ◽  
Before And After ◽  
Centrifugation Technique

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

scholarly journals Surface-area cycling of different surfactant preparations: SP-A and SP-B are essential for large-aggregate integrity

1994 ◽  
Vol 300 (2) ◽  
pp. 519-524 ◽  
Author(s):  
R A Veldhuizen ◽  
S A Hearn ◽  
J F Lewis ◽  
F Possmayer
Keyword(s):  
Surface Area ◽  
Lipid Extract ◽  
Large Aggregate ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Natural Surfactant ◽  
Before And After ◽  
Lipid Adsorption ◽  
Associated Proteins ◽  
Surfactant Phospholipids

Surface-area cycling is an in vitro procedure for the conversion of large into small surfactant aggregates. In this procedure a tube containing a surfactant suspension is rotated end-over-end at 37 degrees C so that the surface area of the suspension changes twice each cycle. We have utilized this method to study the mechanisms involved in aggregate conversion. Several different surfactant preparations were analysed: (1) bovine natural surfactant, a sucrose-gradient-purified material containing surfactant phospholipid and surfactant-associated proteins (SP-) SP-A, SP-B and SP-C; (2) bovine lipid-extract surfactant, which contains the surfactant phospholipids and SP-B and SP-C; (3) mixtures of dipalmitoyl phosphatidylcholine and phosphatidylglycerol (7:3, w/w) reconstituted with one or more surfactant proteins. Aggregate conversion was measured by phosphorus analysis of a 40,000 g supernatant (small aggregate) and pellet (large aggregates) before and after surface-area cycling. Surface-area cycling of lipid extract surfactant or lipids plus SP-B or SP-C resulted in rapid aggregate conversion. Lipids alone were not converted. Only a small percentage of purified natural surfactant was converted into small aggregates. Addition of SP-A to lipid extract surfactant could inhibit aggregate conversion of this material, but this was only observed when an additional 1% (w/w) of SP-B was added to the lipid extract. It is concluded that SP-A is important for large-aggregate integrity. It appears that SP-A acts in conjunction with SP-B. The presence of SP-B and/or SP-C is required for aggregate conversion; it is proposed that this reflects the necessity for lipid adsorption in aggregate conversion.

048. FUNCTIONAL DIFFERENCES BETWEEN SEX-SORTED AND NON-SORTED SPERM

2009 ◽  
Vol 21 (9) ◽  
pp. 13
Author(s):  
G. Evans ◽  
S. P. De Graaf ◽  
W. M.C. Maxwell
Keyword(s):  
Reproductive Tract ◽  
Intrauterine Insemination ◽  
Female Reproductive Tract ◽  
High Fertility ◽  
Bull Semen ◽  
Recent Developments ◽  
Flow Cytometric Sorting ◽  
Ram Sperm

The development and application of flow cytometric sorting for the pre-selection of sex has progressed at an increasing rate since the first report of live pre-sexed offspring of rabbits (2). The technique has been extended to production of pre-sexed offspring of numerous species and sorted bull semen is now widely available commercially around the world. Due to the stresses involved in the sex-sorting process, sex-sorted sperm may be functionally compromised in terms of reduced motility and viability, and their fertilising lifespan within the female reproductive tract may be reduced. Consequently, fertility in vivo may be compromised. However, improvements to the technology and a greater understanding of its biological impact on the sperm have facilitated recent developments in sheep, and we have demonstrated that sex-sorting is capable of selecting a functionally superior ram sperm population in terms of both in vitro and in vivo function. This has resulted in high fertility after intrauterine insemination of sex-sorted ram sperm (1). Unfortunately, to date, these results have not been matched in other species.

Effect of adding heterologous versus homologous bovine seminal plasma prior to cryopreservation on bull sperm quality after thawing

Zygote ◽  
2018 ◽  
Vol 26 (5) ◽  
pp. 388-394
Author(s):  
Thanapol Nongbua ◽  
Essraa M Al-Essawe ◽  
Anders Edman ◽  
Anders Johannisson ◽  
Jane M Morrell
Keyword(s):  
Reactive Oxygen Species ◽  
Seminal Plasma ◽  
Deleterious Effect ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Low Fertility ◽  
Computer Assisted ◽  
High Fertility ◽  
Oxygen Species

SummaryThe aim of this study was to investigate the effect of adding homologous or heterologous bovine seminal plasma (SP) to SP-free sperm samples before freezing on sperm quality after thawing. Ejaculates from bulls of known fertility were used as a source of SP. The SP was removed from further aliquots of the same ejaculates by colloid centrifugation to create SP-free sperm samples; the resuspended sperm pellets were treated with homologous or heterologous SP from high or low fertility bulls at 0%, 1% or 5% before freezing. After thawing, sperm quality was evaluated by computer-assisted sperm analysis and flow cytometry for membrane integrity, reactive oxygen species, chromatin structure, mitochondrial membrane potential and protein tyrosine phosphorylation. Data were analysed using Proc MIXED, SAS®. Post-hoc comparisons were adjusted for multiplicity using Tukey’s method. The addition of SP resulted in significant differences in sperm quality, namely velocity class A, Velocity Straight Line (VSL), Velocity Average Path (VAP), Velocity Curved Line (VCL), Amplitude of Lateral Head Displacement (ALH), Hyperactive (HYP), reactive oxygen species (ROS) production and % DNA fragmentation index (DFI) (P<0.05 for each). Although adding 5% homologous SP from high fertility bulls was beneficial to sperm kinematics, 5% heterologous SP from high fertility bulls had a deleterious effect on chromatin integrity and on sperm velocity. In conclusion, adding SP may have either a beneficial effect or a deleterious effect depending on the individuals involved. It might be feasible to use this method to improve sperm quality in some circumstances.

175 Analysis of Cryopreserved Semen Quality: With the Tools Available, What Is Possible to Be Interpreted

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 112-113
Author(s):  
André F de Andrade
Keyword(s):  
Seminal Plasma ◽  
Semen Quality ◽  
Semen Analysis ◽  
Research Work ◽  
Low Fertility ◽  
Future Research ◽  
Freezing Process ◽  
High Fertility ◽  
Frozen Semen ◽  
Fertility Potential

Abstract Identifying doses of post-thawed semen with high and low fertility potential is the main objective pursued by professionals and companies involved in commercializing frozen semen. In post-thaw semen, we can evaluate the motility characteristics and the integrity of the spermatozoa membranes and their DNA so that we can identify the number of cells that have the minimum characteristics to be considered with the potential to fertilize the oocyte. However, even with these analyzes using fluorescent probes, flow cytometry, and a computerized semen analysis system, it is impossible to predict the dose’s fertilizing potential accurately. The spermatozoa and seminal plasma originate from an individual (e.g., boar, bull, stallion), and even the spermatozoa and seminal plasma from this individual are different between different ejaculates. Factors such as genetics, age, ambiance, nutrition, and semen manipulation can alter the cryotolerance capacity of a given ejaculate, thus affecting its fertility potential. However, recent studies with assessments of proteomics, lipidomics, metabolomics, and miRNAs have associated cryotolerance and semen fertility with markers that can be evaluated before the ejaculate cryopreservation process—in this way, creating the possibility of selecting high fertility doses before the freezing process. Suppose these biological markers will conclusively make it possible to inform whether post-thawed semen dose has high or low fertility potential. In that case, only future research work verifying fertility will allow us to know. In the meantime, it is highly recommended to evaluate the post-thaw semen by assessing characteristics of motility, the integrity of the plasma and acrosomal membranes, and the integrity of DNA. Thus, ensuring that inseminations are carried out with the minimum number of sperm able to provide a high potential for fertility. These minimum numbers are related to the species and type of cryopreserved semen: conventional or sexed.

Sign in / Sign up

Export Citation Format

Share Document