Stallion Sperm Viability, as Measured by the Nucleocounter SP-100, Is Affected by Extender and Enhanced by Single Layer Centrifugation

On-stud assessment of stallion sperm quality can be problematic. A new instrument, the Nucleocounter SP-100, was validated for measuring stallion sperm concentration and viability. It was subsequently used to evaluate sperm viability in Kenney's extender and INRA96. There was a strong correlation between sperm concentrations measured by the Nucleocounter SP-100 and by the Bürker counting chamber (; ). Similarly, there was a good correlation between sperm viability results from the Nucleocounter SP-100 and flow cytometric results (; ). Sperm viability at 24 hours was significantly better for samples extended in INRA96 than in Kenney's extender (). Furthermore, sperm kinematics were better for stored samples in INRA96 than in Kenney's extender. Single Layer Centrifugation selected spermatozoa that maintained their viability better during storage for 24 hours than the uncentrifuged samples. In conclusion, the type of semen extender used and Single Layer Centrifugation were found to influence both the kinematics and viability of stallion spermatozoa. The Nucelocounter-SP100 was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability.

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Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration

Reproduction Fertility and Development ◽

10.1071/rd19154 ◽

2020 ◽

Vol 32 (9) ◽

pp. 873

Author(s):

Yue Liu ◽

Megan Chesnut ◽

Amy Guitreau ◽

Jacob Beckham ◽

Adam Melvin ◽

...

Keyword(s):

Sperm Quality ◽

Low Cost ◽

Sperm Concentration ◽

Single Layer ◽

Control Device ◽

Cell Counts ◽

Counting Chamber ◽

Product Costs ◽

Base Platform ◽

Microfabrication Techniques

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 μm×100 μm) and a PDMS base platform to create a known volume with a 10-μm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations.

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54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab54 ◽

2017 ◽

Vol 29 (1) ◽

pp. 134

Author(s):

T. Nongbua ◽

A. Utta ◽

N. Am-In ◽

J. Suwimonteerabutr ◽

A. Johannisson ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Chromatin Structure ◽

Mitochondrial Membrane ◽

Linear Mixed Model ◽

Sperm Quality ◽

Single Layer ◽

Sperm Chromatin ◽

Sperm Viability ◽

Bull Sperm

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

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14 Sperm membrane integrity in ejaculates from young bulls may be improved by single-layer centrifugation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab14 ◽

2019 ◽

Vol 31 (1) ◽

pp. 133

Author(s):

E. Hurri ◽

A. Johannisson ◽

I. Lim-Verde ◽

J. M. Morrell

Keyword(s):

Sperm Quality ◽

Pearson Correlation ◽

Animal Health ◽

Sperm Concentration ◽

Membrane Integrity ◽

Single Layer ◽

Before And After ◽

Sperm Membrane ◽

Chromatin Integrity ◽

Species Production

Sperm quality in the first ejaculates of young bulls is often poor, leading to rejection of these samples for commercial freezing. It may be several weeks before sperm quality reaches the thresholds regarded as being acceptable for commercial production. Single-layer centrifugation (SLC) has been shown to separate robust bull spermatozoa from the rest of the ejaculate (Goodla et al. 2015 J. Dairy Sci. 97, 2204-2212; Nongbua et al. 2017 Reprod Domest. Anim. 52, 596-602). The aim of this study was to test whether SLC can be used to select spermatozoa from the ejaculates of young bulls and thus freeze samples earlier than is currently possible. Ejaculates (at least 3 per bull; n=33) were collected from 9 young Holstein or Swedish Red bulls, 255 to 415 days old. The sperm concentration was adjusted to 69×106 spermatozoa mL−1 with Andromed™ (Minitub, Tiefenbach, Germany) before layering part of the sample over 4mL of Bovicoll colloid. The remaining sample served as the unselected control. After centrifugation at 300×g for 20min, the sperm pellet was aspirated into fresh extender. Both control (uncentrifuged) and SLC samples were frozen and stored in LN pending analysis. Membrane integrity was evaluated by flow cytometry after staining for 10min at 37°C with SYBR14/propidium iodide (Live-Dead Sperm Viability Kit, L-7011; Invitrogen, Carlsbad, CA, USA). Paired t-test was used to compare results for control and SLC samples, and Pearson correlation was used for age and membrane integrity. The median age at which the samples were taken was 319 days (range: 255-415 days). Membrane integrity varied considerably among bulls (range: 11-72%). The mean proportion of membrane-intact spermatozoa in controls and SLC samples was 40±15 and 47±16%, respectively (P<0.01). In 21 of the 33 ejaculates (64%), the SLC sample had a higher membrane integrity than the controls (Table 1). Age in days was positively correlated with membrane integrity for SLC samples (r=0.40; P<0.05) but not for controls. These results suggest that SLC might be a useful technique for selecting membrane-intact spermatozoa from the ejaculates of young bulls. However, there is considerable variation among bulls, possibly due to age, which is currently being investigated. Other parameters of sperm quality, such as chromatin integrity, mitochondrial membrane potential reactive oxygen species production, and motility, are also being evaluated. Table 1.Age and membrane integrity (mean±standard deviation) in ejaculates from young bulls before and after SLC (n=33) This project was funded by the Royal Swedish Academy of Agriculture and Forestry, Viking Genetics, and the Faculty for Veterinary Medicine and Animal Health, SLU. We thank Viking Genetics for supplying the semen samples.

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Effects of Dietary Supplementation of Conjugated Linoleic Acids and Their Inclusion in Semen Extenders on Bovine Sperm Quality

Animals ◽

10.3390/ani11020483 ◽

2021 ◽

Vol 11 (2) ◽

pp. 483

Author(s):

Mohammed S. Liman ◽

Vittoria Franco ◽

Claudia L. Cardoso ◽

Valentina Longobardi ◽

Bianca Gasparrini ◽

...

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Sperm Quality ◽

Sperm Concentration ◽

Conjugated Linoleic Acids ◽

Factor I ◽

Bovine Sperm ◽

Igf I ◽

Semen Extender ◽

Oxidative Species

Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks −2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates’ total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.

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Improvement of cryopreservation protocol in both purebred horses including Spanish horses

Spanish Journal of Agricultural Research ◽

10.5424/sjar/2018164-13677 ◽

2019 ◽

Vol 16 (4) ◽

pp. e0406

Author(s):

Jordi Miró ◽

Marion Papas

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Observation Point ◽

Sperm Cells ◽

Sperm Viability ◽

Dilution Ratio ◽

Sperm Cryopreservation ◽

Morphological Abnormalities ◽

Cryopreservation Protocol

There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio.

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Glutathione supplementation in semen extender improves rabbit sperm attributes during refrigeration

World Rabbit Science ◽

10.4995/wrs.2021.14759 ◽

2021 ◽

Vol 29 (2) ◽

pp. 81

Author(s):

Ejaz Ahmad ◽

Zahid Naseer ◽

Melih Aksoy

Keyword(s):

Sperm Motility ◽

Acrosome Reaction ◽

Reaction Rate ◽

Sperm Quality ◽

Final Concentration ◽

Sperm Viability ◽

Acrosome Integrity ◽

Semen Extender ◽

Sperm Motion ◽

Artificial Vagina

In the present study, we evaluated the sustaining effect of various glutathione (GSH) concentrations in extender on rabbit sperm attributes during storage at 5°C for 24 h. Semen was collected from regular donor rabbit bucks using an artificial vagina and initially evaluated for sperm quality. The qualifying ejaculates were diluted with one of the extenders having 0, 1, 2, 4 or 8 mM GSH, to achieve a final concentration of 1×108 sperm/mL. The extended samples were stored at 5°C for 24 h. Sperm motility, motion kinetics, acrosome integrity and viability were assessed after 3, 6, 12 and 24 h of storage. The results showed that total sperm motility and sperm motion kinetics (oscillation index of the sperm, straightness index and beat cross frequency) were influenced (<em>P</em><0.05) by glutathione dose and refrigeration time. An interaction of (<em>P</em><0.05) GSH concentrations and refrigeration time was observed for sperm viability and acrosome reaction rate. In conclusion, the 4 mM GSH supplemented extender’s protective influence was remarkable to maintainrabbit sperm quality for 24 h 5°C.

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Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation

Antibiotics ◽

10.3390/antibiotics7010001 ◽

2017 ◽

Vol 7 (1) ◽

pp. 1 ◽

Cited By ~ 4

Author(s):

Ziyad Al-Kass ◽

Joachim Spergser ◽

Christine Aurich ◽

Juliane Kuhl ◽

Kathrin Schmidt ◽

...

Keyword(s):

Sperm Quality ◽

Single Layer ◽

Semen Extender

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A diluent containing coconut water, fructose, and chicken egg yolk increases rooster sperm quality at 5°C

Veterinary World ◽

10.14202/vetworld.2019.1116-1120 ◽

2019 ◽

Vol 12 (7) ◽

pp. 1116-1120 ◽

Cited By ~ 2

Author(s):

Siti Eliana Rochmi ◽

Miyayu Soneta Sofyan

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Egg Yolk ◽

Mean Value ◽

Coconut Water ◽

Sperm Viability ◽

Spermatozoa Motility ◽

Chicken Egg ◽

Semen Volume ◽

Chicken Egg Yolk

Aim: The present study was conducted to evaluate the quality of rooster sperm at 5°C after treatment with a diluent containing coconut water, fructose, and chicken egg yolk and stored the semen sample at 5°C. Materials and Methods: Ten semen samples from 10 healthy roosters were subjected to four different treatments. For the treatments, 0.2 ml fresh semen with a sperm concentration of 5.2X109 cell/ml was mixed with T0 (no diluent), T1 (0.34 ml coconut water and 6 μl fructose), T2 (0.274 ml coconut water, 0.12 ml egg yolk, and 6 μl fructose), and T3 (0.34 ml egg yolk and 6 μl fructose) solutions. Each treated solution was stored at 5°C and evaluated both macroscopically and microscopically. Macroscopically, semen volume, pH, and sperm concentration were evaluated. The microscopic sperm characteristics examined included total motility (i.e., rapid, medium, or slow), progressive and non-progressive motility, viability, and spermatozoa abnormalities noted at different storage times. The results showed that spermatozoa motility was under 40%. Results: The results indicated that sperm viability significantly affected (p<0.05). The highest mean value of sperm viability on day 7 of storage was found after treatment with the T2 solution (46.100±0.5677%). Similarly, spermatozoa abnormalities were significantly lower after treatment with the T2 solution (6.680±1.702%). Conclusion: The addition of a diluent containing coconut water, egg yolk, and fructose helped in the better preservation spermatozoa motility, as well as viability for up to 7 days when the semen samples were stored at 5°C.

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Effects of Double Layer Centrifugation on the Improvement of Sperm Quality in Dogs: A Comparative Note among Different Breeds

Annals of Veterinary Science ◽

10.31829/2691-5502/avs2021-4(1)-003 ◽

2021 ◽

pp. 1-12

Keyword(s):

Double Layer ◽

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Sperm Concentration ◽

Single Layer ◽

Progressive Motility ◽

Prolonged Contact ◽

And Storage ◽

Fresh Semen

The evaluation of sperm quality in the laboratory is essential to improve efﬁciency in assisted reproduction. As in other species, for the dog there are reports that prolonged contact of sperm with some components of seminal plasma is associated with decreased motility and sperm viability. Thus, the centrifugation is a technique widely used to concentrate the spermatozoa and eliminate the supernatant. The purpose of this study was to evaluate the effect of double layer centrifugation on the percentages of total sperm motility and progressive sperm motility of the dog’s semen submitted to the dilution, single layer centrifugation, cooling and storage at 5 °C for 24 and 48 hours. For this purpose, ejaculates of 30 healthy male dogs were evaluated, by taking into account the comparison among the conventional sperm parameters (ejaculate volume, sperm concentration, total sperm motility and sperm progressive motility). The semen samples were examined in standard baseline condition of fresh semen (FS), after dilution (AD), after dilution and single layer centrifugation (SLC), after double layer centrifugation (DLC). According to the different time points, the semen samples were evaluated in baseline conditions, immediately after their collection at (T0), at 24 h (T24) and at 48 h (T48), to evaluate the effect of different treatments on the semen’s quality. Results showed a significant effect of double layer centrifugation on the improvement of total sperm motility and progressive sperm motility percentages of dogs. The use of cooling fresh semen soon after the double layer centrifugation will improve the semen quality up to 48h, with a special emphasis for the percentages of total sperm motility and sperm progressive motility, adding an alternative technical approach to reproductive performance in male breeding dogs.

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Qiangjing Tablets Regulate Apoptosis and Oxidative Stress via Keap/Nrf2 Pathway to Improve the Reproductive Function in Asthenospermia Rats

Frontiers in Pharmacology ◽

10.3389/fphar.2021.714892 ◽

2021 ◽

Vol 12 ◽

Author(s):

Guangsen Li ◽

Peihai Zhang ◽

Yaodong You ◽

Diang Chen ◽

Jian Cai ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Potential ◽

Luteinizing Hormone ◽

Western Blot ◽

Mitochondrial Membrane ◽

Sperm Quality ◽

Sperm Concentration ◽

Reproductive Function ◽

Sperm Viability ◽

And Oxidative Stress

Asthenozoospermia (AZS), is a common cause of male infertility. Currently, most drugs for azoospermia lack desirable therapeutic efficiency, therefore developing new drug therapy is important. Qiangjing tablets could enhance renal function and improve sperm quality. The purpose of this study was to examine whether Qiangjing tablets could improve the reproductive function in azoospermia rats through activating the Nrf2/ARE pathway, and how to regulate energy metabolism and oxidative stress in this process. Sperm motility, sperm concentration and sperm viability were detected by WLJY-9000 Weili Digital Color Sperm Quality Detection System. HE staining was used to observe the pathological condition of testis in AZS rats. Cell apoptosis was analyzed by Tunnel staining and flow cytometry. The changes of mitochondrial membrane potential were detected by JC-1. The levels of Estradiol, testosterone and luteinizing hormone, activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and content of malondialdehyde (MDA) and glutathione (GSH) were detected by ELISA. The effects of Qiangjing Tablets on GC-1 spgs and Nrf2 protein were investigated through CCK-8 assay and western blot. The expression levels of HO-1, Keap1, and P-Nrf2 were detected by western blot. The results demonstrated that Qiangjing tablets upregulated levels of sperm motility, sperm concentration and sperm viability, which was shown to significantly increase levels of HO-1, Keap1, P-Nrf2, Estradiol and testosterone, along with increasing the activity of SOD, GSH-Px and GSH and suppressing the MDA content, luteinizing hormone and Vimentin level. Qiangjing tablets could significantly inhibit spermatogenic cells apoptosis and promote GC-1 spgs viability, increase PE/FITC ratio, mitochondrial membrane potential and reduc oxidative stress. Qiangjing tablets protected spermatogenic cell to upregulate male sex hormoneto, improved the sperm quality and reproductive function in AZS rats via activating the Keap/Nrf2 signaling pathway.

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