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Keywords = spermatozoa subpopulations

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13 pages, 1035 KiB  
Article
The Distribution of Boars Spermatozoa in Morphometrically Distinct Subpopulations after In Vitro Exposure to Radiofrequency Electromagnetic Radiation at 2500 MHz and Their Motility
by Ivona Žura Žaja, Silvijo Vince, Ivan Butković, Kim Senaši, Nina Poljičak Milas, Krešimir Malarić, Martina Lojkić, Ivan Folnožić, Suzana Milinković Tur, Mario Kreszinger, Marko Samardžija, Snježana Čipčić, Nikolino Žura, Mario Ostović and Marinko Vilić
Animals 2024, 14(6), 828; https://doi.org/10.3390/ani14060828 - 7 Mar 2024
Cited by 3 | Viewed by 1457
Abstract
Anthropogenic radiofrequency electromagnetic radiation (RF-EMR) from wireless technologies has increased dramatically. The boar semen used for artificial insemination is essential in sustaining the pig industry, and additionally it is also exposed to the effects of the RF-EMR of wireless technologies. Furthermore, there are [...] Read more.
Anthropogenic radiofrequency electromagnetic radiation (RF-EMR) from wireless technologies has increased dramatically. The boar semen used for artificial insemination is essential in sustaining the pig industry, and additionally it is also exposed to the effects of the RF-EMR of wireless technologies. Furthermore, there are no data on the effects of RF-EMR on semen quality, and this is the first analysis of sperm’s morphometric parameters for assessing the effect of RF-EMR on the spermatozoa subpopulations of boars. This study investigated the effect of RF-EMR on in vitro exposed breeding boar semen spermatozoa motility and the proportions of spermatozoa subpopulations according to their morphometric head and tail parameters. The semen samples of 12 boars were divided into control and experimental groups. The samples in the experimental group were exposed in a gigahertz transverse electromagnetic chamber at a frequency of 2500 MHz (the frequency band used in 5G technology) and an electric field strength of 10 Vm−1 for two hours. After exposure, the spermatozoa motility was evaluated for both groups. A morphometric analysis of the semen smears was performed using SFORM software (Version 1.0; VAMS, Zagreb, Croatia). The progressive spermatozoa motility was significantly reduced in the experimental group (74.7% vs. 85.7%). PC analysis and cluster analysis revealed two spermatozoa subpopulations: S1, spermatozoa with a more regular head shape and a smaller midpiece outline, and S2, spermatozoa with a more elongated head shape and a larger midpiece outline. The experimental semen samples had a greater proportion of the S1 spermatozoa subpopulation (68.2% vs. 64.4%). The effect of RF-EMR at 2500 MHz on the in vitro exposed boar semen resulted in decreased progressive spermatozoa motility and a lower proportion of the spermatozoa subpopulation with a higher fertilizing potential. Full article
(This article belongs to the Special Issue Animal Reproduction: Semen Quality Assessment, Volume II)
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14 pages, 331 KiB  
Article
Kinematic and Morphometric Assessment of Fresh Semen, before, during and after Mating Period in Brahman Bulls
by Ignacio Araya-Zúñiga, Francisco Sevilla, Rafael Molina-Montero, Eduardo R. S. Roldan, Manuel Barrientos-Morales, Miguel A. Silvestre and Anthony Valverde
Animals 2024, 14(1), 132; https://doi.org/10.3390/ani14010132 - 29 Dec 2023
Cited by 4 | Viewed by 2949
Abstract
The objective of the present study was to determine the effects that the reproductive season has on the motility, kinematics, morphology, and sperm morphometry of Brahman bulls evaluated with a commercial CASA system. The experiment was carried out at the Costa Rica Institute [...] Read more.
The objective of the present study was to determine the effects that the reproductive season has on the motility, kinematics, morphology, and sperm morphometry of Brahman bulls evaluated with a commercial CASA system. The experiment was carried out at the Costa Rica Institute of Technology from March to August 2021. A total of eight Brahman bulls were used. A total of 28 ejaculates were collected in the pre-mating period (PMP), during it (DMP), and after it (AMP) using an electroejaculator. The sperm concentration was measured with the Accuread photometer. The motility was measured using a Spermtrack® counting chamber. The analyses were performed with the CASA-Mot ISAS®v1 system. The morphology was analyzed using a microscope with a negative phase contrast objective. Morphometry was evaluated with the CASA-Morph. The sperm concentration did not present differences between the PMP and AMP; however, it was significantly higher than DMP (p > 0.05). Regarding the progressiveness variables, linearity on forward progression (LIN), straightness (STR), and wobble (WOB) were higher (p < 0.05) DMP. A kinematic principal component analysis grouped all the variables into three factors and an effect on the reproductive period was found (p < 0.05) in the parameters of the head and middle part of the sperm, such as width and perimeter, which were greater in the PMP. The length of the sperm head in the PMP and DMP did not show differences; however, both were larger (p < 0.05) than AMP. The insertion distance of the middle piece of the sperm was significantly greater than DMP. Finally, the PMP contained cells with a larger insertion angle (p < 0.05) than AMP. These findings are important to understand the implications of reproductive status on sperm quality and to consider them in andrological evaluations. Full article
(This article belongs to the Section Animal Reproduction)
14 pages, 1771 KiB  
Article
Motility Subpopulations with Distinct Motility Characteristics Using Swim-Up-Selected Sperm Cells from Norwegian Red Bulls: Effects of Freezing–Thawing and Between-Bull Variation
by Anne Hege Alm-Kristiansen
Biology 2023, 12(8), 1086; https://doi.org/10.3390/biology12081086 - 3 Aug 2023
Cited by 5 | Viewed by 1920
Abstract
Discrete subpopulations of motile sperm cells have been found for several species and are implicated to be important for sperm functionality. The aim of this present study was to examine the motile subpopulations in swim-up-selected bull spermatozoa and the relationship between subpopulations in [...] Read more.
Discrete subpopulations of motile sperm cells have been found for several species and are implicated to be important for sperm functionality. The aim of this present study was to examine the motile subpopulations in swim-up-selected bull spermatozoa and the relationship between subpopulations in fresh and frozen–thawed sperm cells. In experiment 1, swim-up (SWUP)-selected and non-selected (control) sperm cells were analyzed using a Computer-Assisted Sperm Analyzer (CASA). In experiment 2, the semen from nine bulls was cryopreserved and analyzed using CASA both before and after freezing and after incubation at physiological temperatures. The SWUP population had a higher proportion of total motility, progressivity, and velocity compared to the control (p < 0.05). Likewise, both incubation over time and cryopreservation affected motility and motility parameters (p < 0.05). The population of rapid progressive (RapidP) sperm cells dominated the SWUP fraction and was higher than in the control samples (p < 0.05). Furthermore, RapidP was also the main part of fresh semen, but decreased significantly over time during incubation and due to cryopreservation. In conclusion, RapidP was the main population in SWUP-selected spermatozoa and seems to be an important subpopulation contributing to the differences between treatments and in response to the freezing of sperm cells. Full article
(This article belongs to the Section Developmental and Reproductive Biology)
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11 pages, 1627 KiB  
Article
ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen–Thawed Horse Semen
by Marta Dordas-Perpinyà, Ivan Yanez-Ortiz, Nicolas Sergeant, Vincent Mevel, Jean-François Bruyas, Jaime Catalán, Maryse Delehedde, Lamia Briand-Amirat and Jordi Miró
Animals 2022, 12(23), 3417; https://doi.org/10.3390/ani12233417 - 4 Dec 2022
Cited by 13 | Viewed by 3191
Abstract
ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and [...] Read more.
ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06–35.61 ng/10M spz; n = 23) or high (39.92–82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen. Full article
(This article belongs to the Special Issue Sperm Quality and Fertility of Livestock Animals)
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2 pages, 205 KiB  
Abstract
Sperm Characterization of Endangered Leuciscids Endemic from the Iberian Peninsula: Gamete Storage as a Tool for Helping Ex-Situ Breeding Programs
by Ana Hernández, Carla Sousa-Santos, Fátima Gil, Pedro M. Guerreiro, Elsa Cabrita and Victor Gallego
Biol. Life Sci. Forum 2022, 13(1), 140; https://doi.org/10.3390/blsf2022013140 - 20 Jun 2022
Viewed by 1590
Abstract
Populations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and several types of actions (from in situ to ex situ measurements) have been applied over the past decades for preserving these species. However, limited knowledge [...] Read more.
Populations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and several types of actions (from in situ to ex situ measurements) have been applied over the past decades for preserving these species. However, limited knowledge about their reproductive biology makes it necessary to investigate different aspects of the reproductive cycle for improving breeding programs. The main objectives of this work were to advance knowledge concerning sperm kinetics and spermatozoa morphology and to develop protocols for the short- and long- term storage of gametes. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum, Achondrostoma occidentale, and Squalius aradensis) were sampled during the spring of 2022 both in captive populations kept at Aquário Vasco da Gama (AVG) and in wild populations from different Portuguese rivers. Sperm samples were collected and sperm motion parameters were assessed, for the first time, for these four species. Sperm kinetics differed between species in motility and velocity traits, also showing a different number of sperm subpopulations. The longevity of sperm (swimming period) was also different among species: the shortest period was obtained for the wild population of S. aradensis (values close to zero at 40 s), and the longest swimming period for the captive population of I. lusitanicum (values close to zero at 120 s). Furthermore, different storage trials were carried out diluting the sperm in a extender solution (75 mM NaC1, 70 mM KC1, 2 mM CaC12, 1 mM, MgSO4, l0 mM Hepes, pH 8) at a ratio 1:20 (sperm:extender). Sperm quality (>40% of motile cells) was kept for a maximum of four days of storage, depending on the species. In addition, new cryopreservation protocols (using DMSO, Methanol and/or egg yolk) were tested for cryobanking the sperm of these threatened species. Cryopreserved samples showed significantly lower motility when compared with fresh samples, and the best results were obtained for I. lusitanicum, reaching 20% of motile cells after thawing using 10% of DMSO supplemented with 10% of egg yolk. This study is the first of its kind to successfully achieve gamete cryopreservation of Iberian endemic and endangered freshwater fish species, developing new and useful tools to complement the management and conservation programs. Full article
(This article belongs to the Proceedings of The IX Iberian Congress of Ichthyology)
22 pages, 3446 KiB  
Article
Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males
by Marta Olszewska, Oliwia Kordyl, Marzena Kamieniczna, Monika Fraczek, Piotr Jędrzejczak and Maciej Kurpisz
Int. J. Mol. Sci. 2022, 23(9), 4516; https://doi.org/10.3390/ijms23094516 - 19 Apr 2022
Cited by 5 | Viewed by 3489
Abstract
Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm [...] Read more.
Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure. Full article
(This article belongs to the Special Issue Molecular Biology of Human Fertility)
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13 pages, 1046 KiB  
Article
Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs
by Guillaume Domain, Hiba Ali Hassan, Eline Wydooghe, Osvaldo Bogado Pascottini, Anders Johannisson, Jane M. Morrell, Wojciech Niżański and Ann Van Soom
Animals 2022, 12(6), 714; https://doi.org/10.3390/ani12060714 - 11 Mar 2022
Cited by 8 | Viewed by 3045
Abstract
This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) ≥ 80%, n = 9; suboptimal: NM [...] Read more.
This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) ≥ 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile. Full article
(This article belongs to the Special Issue Biotechnology and Reproduction in Companion Animals)
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17 pages, 1836 KiB  
Article
Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions
by Patricia Peris-Frau, Irene Sánchez-Ajofrín, Alicia Martín Maestro, Carolina Maside, Daniela Alejandra Medina-Chávez, Olga García-Álvarez, María del Rocío Fernández-Santos, Vidal Montoro, José Julián Garde, Manuel Ramón and Ana Josefa Soler
Biology 2021, 10(11), 1213; https://doi.org/10.3390/biology10111213 - 20 Nov 2021
Cited by 8 | Viewed by 2827
Abstract
The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, [...] Read more.
The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions. Full article
(This article belongs to the Special Issue Factors Affecting In Vitro Assessment of Sperm Quality)
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10 pages, 869 KiB  
Article
Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls
by Jesús L. Yániz, Inmaculada Palacín, Miguel A. Silvestre, Carlos Olegario Hidalgo, Carolina Tamargo and Pilar Santolaria
Biology 2021, 10(11), 1135; https://doi.org/10.3390/biology10111135 - 4 Nov 2021
Cited by 7 | Viewed by 2659
Abstract
The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls [...] Read more.
The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests. Full article
(This article belongs to the Special Issue Sperm Quality: Past, Present and the Future Knowledge We Need)
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13 pages, 856 KiB  
Article
Effects of Three Semen Extenders, Breeding Season Month and Freezing–Thawing Cycle on Spermatozoa Preservation of Portuguese Merino Sheep
by Margarida Fernandes, Pablo Rodríguez Hernández, João Simões and João Pedro Barbas
Animals 2021, 11(9), 2619; https://doi.org/10.3390/ani11092619 - 7 Sep 2021
Cited by 13 | Viewed by 4514
Abstract
This study aimed to evaluate and compare the effect of three semen extenders (S-EXT) on 22 spermatozoa (SPZ) parameters (subjective and computer-assisted sperm analysis evaluations), before and after semen cryopreservation throughout different months of the breeding season in the Portuguese Merino breed. According [...] Read more.
This study aimed to evaluate and compare the effect of three semen extenders (S-EXT) on 22 spermatozoa (SPZ) parameters (subjective and computer-assisted sperm analysis evaluations), before and after semen cryopreservation throughout different months of the breeding season in the Portuguese Merino breed. According to the multivariable model, the SPZ viability (alive %), kinetics subjective individual motility, total motility, total progressive motility and its subpopulations, and beat cross frequency) were higher in the egg yolk-based S-EXT improved by Estação Zootécnica National (Portugal) than in Ovixcell® or Andromed® extenders. All the differences were only observed in thawed semen, except for total motility and total progressive motility, in which Ovixcell® also showed the poorest results on fresh semen. An interaction effect between S-EXT and semen processing was observed on 72.3% (17/22) of the evaluated parameters, evidencing a variable cryoprotective action between S-EXT. The SPZ viability was poorer in the onset of the breeding season (end of April/early May) than in the previous middle breeding season (November/early December), suggesting the influence of a short anoestrous season on ejaculate quality, even though the volume and SPZ concentration of the ejaculates remained stable throughout the experiment. Additionally, S-EXT x semen processing x month interaction effect on 59.1% (13/22) of the evaluated parameters evidenced the importance of SPZ time collection in a natural environment to cryopreserve ram’s semen. We concluded that, overall, the egg yolk-based S-EXT provided a greater value to the cryopreservation of Merino rams´ semen. Nevertheless, the causes of the interaction effect between S-EXT, semen processing and/or month on several SPZ parameters should be addressed, including SPZ molecular research in new studies, in order to improve egg yolk-based as well as in egg yolk-free-based S-EXT. Full article
(This article belongs to the Special Issue Reproductive Physiology of Small Ruminants)
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15 pages, 975 KiB  
Article
Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination
by Vinicio Barquero, Eduardo R. S. Roldan, Carles Soler, Jesús L. Yániz, Marlen Camacho and Anthony Valverde
Animals 2021, 11(4), 920; https://doi.org/10.3390/ani11040920 - 24 Mar 2021
Cited by 24 | Viewed by 3989
Abstract
The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows [...] Read more.
The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows produced from crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11 sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormal sperm were used for artificial inseminations (AI) and included in the study. Samples were analyzed using an ISAS®v1 computer-assisted sperm analysis system for eight morphometric parameters of head shape and size (CASA-Morph). Sub-populations of morphometric ejaculates were characterized using multivariate procedures, such as principal component (PC) analysis and clustering methods (k-means model). Four different ejaculate sub-populations were identified from two PCs that involved the head shape and size of the spermatozoa. The discriminant ability of the different morphometric sperm variables to predict sow litter size was analyzed using a receiver operating characteristics (ROC) curve analysis. Sperm head length, ellipticity, elongation, and regularity showed significant predictive capacity on litter size (0.59, 0.59, 0.60, and 0.56 area under curve (AUC), respectively). The morphometric sperm sub-populations were not related to sow litter size. Full article
(This article belongs to the Special Issue Biotechnologies Applied to Animal Reproduction)
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18 pages, 2188 KiB  
Article
Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation
by Filipa Bubenickova, Pavla Postlerova, Ondrej Simonik, Jitka Sirohi and Jiri Sichtar
Int. J. Mol. Sci. 2020, 21(17), 6415; https://doi.org/10.3390/ijms21176415 - 3 Sep 2020
Cited by 22 | Viewed by 4202
Abstract
Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. [...] Read more.
Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep−) fractions. The addition of three concentrations—125, 250, and 500 µg/mL—of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep− fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions. Full article
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17 pages, 1789 KiB  
Article
Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure
by Jaime Catalán, Marion Papas, Sabrina Gacem, Yentel Mateo-Otero, Joan E. Rodríguez-Gil, Jordi Miró and Marc Yeste
Biology 2020, 9(9), 254; https://doi.org/10.3390/biology9090254 - 29 Aug 2020
Cited by 12 | Viewed by 3774
Abstract
Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and [...] Read more.
Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects. Full article
(This article belongs to the Section Biotechnology)
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16 pages, 516 KiB  
Article
Kinematic Sub-Populations in Bull Spermatozoa: A Comparison of Classical and Bayesian Approaches
by Luis Víquez, Vinicio Barquero, Carles Soler, Eduardo R.S. Roldan and Anthony Valverde
Biology 2020, 9(6), 138; https://doi.org/10.3390/biology9060138 - 26 Jun 2020
Cited by 30 | Viewed by 5095
Abstract
The ejaculate is heterogenous and sperm sub-populations with different kinematic patterns can be identified in various species. Nevertheless, although these sub-populations are statistically well defined, the statistical differences are not always relevant. The aim of the present study was to characterize kinematic sub-populations [...] Read more.
The ejaculate is heterogenous and sperm sub-populations with different kinematic patterns can be identified in various species. Nevertheless, although these sub-populations are statistically well defined, the statistical differences are not always relevant. The aim of the present study was to characterize kinematic sub-populations in sperm from two bovine species, and diluted with different commercial extenders, and to determine the statistical relevance of sub-populations through Bayesian analysis. Semen from 10 bulls was evaluated after thawing. An ISAS®v1 computer-assisted sperm analysis (CASA)-Mot system was employed with an image acquisition rate of 50 Hz and ISAS®D4C20 counting chambers. Sub-populations of motile spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods (k-means model). Four different sperm sub-populations were identified from three PCs that involved progressiveness, velocity, and cell undulatory movement. The proportions of the different sperm sub-populations varied with the extender used and in the two species. Despite a statistical difference (p < 0.05) between extenders, the Bayesian analysis confirmed that only one of them (Triladyl®) presented relevant differences in kinematic patterns when compared with Tris-EY and OptiXcell®. Extenders differed in the proportion of sperm cells in each of the kinematic sub-populations. Similar patterns were identified in Bos taurus and Bos indicus. Bayesian results indicate that sub-populations SP1, SP2, and SP3 were different for PC criteria and these differences were relevant. For velocity, linearity, and progressiveness, the SP4 did not show a relevant difference regarding the other sperm sub-populations. The classical approach of clustering or sperm subpopulation thus may not have a direct biological meaning. Therefore, the biological relevance of sperm sub-populations needs to be reevaluated. Full article
(This article belongs to the Special Issue Factors Affecting In Vitro Assessment of Sperm Quality)
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20 pages, 3653 KiB  
Article
Melatonin Non-Linearly Modulates Bull Spermatozoa Motility and Physiology in Capacitating and Non-Capacitating Conditions
by Estela Fernández-Alegre, Indira Álvarez-Fernández, Juan Carlos Domínguez, Adriana Casao and Felipe Martínez-Pastor
Int. J. Mol. Sci. 2020, 21(8), 2701; https://doi.org/10.3390/ijms21082701 - 13 Apr 2020
Cited by 11 | Viewed by 3082
Abstract
Bull spermatozoa physiology may be modulated by melatonin. We washed ejaculated spermatozoa free of melatonin and incubated them (4 h, 38 °C) with 0-pM, 1-pM, 100-pM, 10-nM and 1-µM melatonin in TALP-HEPES (non-capacitating) and TALP-HEPES-heparin (capacitating). This range of concentrations encompassed the effects [...] Read more.
Bull spermatozoa physiology may be modulated by melatonin. We washed ejaculated spermatozoa free of melatonin and incubated them (4 h, 38 °C) with 0-pM, 1-pM, 100-pM, 10-nM and 1-µM melatonin in TALP-HEPES (non-capacitating) and TALP-HEPES-heparin (capacitating). This range of concentrations encompassed the effects mediated by melatonin receptors (pM), intracellular targets (nM–µM) or antioxidant activity (µM). Treatment effects were assessed as motility changes by computer-assisted sperm analysis (CASA) of motility and physiological changes by flow cytometry. Melatonin effects were more evident in capacitating conditions, with 100 pM reducing motility and velocity (VCL) while increasing a “slow” subpopulation. All concentrations decreased apoptotic spermatozoa and stimulated mitochondrial activity in viable spermatozoa, with 100 pM–1 µM increasing acrosomal damage, 10 nM–1 µM increasing intracellular calcium and 1 pM reducing the response to a calcium-ionophore challenge. In non-capacitating media, 1 µM increased hyperactivation-related variables and decreased apoptotic spermatozoa; 100 pM–1 µM increased membrane disorders (related to capacitation); all concentrations decreased mitochondrial ROS production. Melatonin concentrations had a modal effect on bull spermatozoa, suggesting a capacitation-modulating role and protective effect at physiological concentrations (pM). Some effects may be of practical use, considering artificial reproductive techniques. Full article
(This article belongs to the Special Issue Advances in Molecular Regulation of Spermatozoa Function)
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