Reproducibility of computer-aided semen analysis: comparison of five different systems used in a practical workshop**Data obtained at a British Andrology Society workshop on Computer-assisted Sperm Motility Analysis at the Royal Veterinary College, London, United Kingdom, September 24 to 25, 1992.

With the growing number of boar studs having semen analysis performed by reproductive specialists, a growing number of diagnostic challenges are encountered. Semen analysis classically involves evaluation of sperm cell motility, morphology, and concentration; however, culture of the extended semen sample for bacterial contamination has become routine. Two isolates, Achromobacter xylosoxidans and Ralstonia pickettii, have recently been identified in the water distillation system of a boar stud facility that uses this water to extend the raw semen in various semen extenders. Insemination of sows with contaminated semen has resulted in severe pyometras diagnosed on necropsy. The effect of these bacteria on sperm motility has not been examined in a controlled setting. The objective of this study was to determine the effects of A. xylosoxidans (AX) and R. pickettii (RP) on pH and motility in culture-negative semen samples over a 7-day period at 16�C. Banked clinical isolates of AX and RP were plated on Columbia blood agar and incubated for 48 h at 37�C. For each isolate, a single colony was selected and transferred to 10 mL of Luria broth. The broth was then incubated for 24 h at 37�C. Optical density measurements were performed at 24 and 48 h of growth, followed by quantification of bacteria by plate counts of serially diluted broth cultures (colony forming units). At 24 h, AX and RP reached levels of 1 � 108 and 1 � 107 [colony-forming units (cfu) mL–1], respectively. Concentration of bacteria in clinical infection was determined to be approximately 1 � 104 and 102 for AX and RP, respectively. In order to attain concentrations similar to those in clinical infection, dilution of the bacteria was necessary. Centrifugation of broth culture at 4000 rpm for 5 min was performed and the bacterial pellet was re-suspended in culture-negative semen in Modena (SGI, LTD, Cambridge, IA, USA) extender to concentrations mimicking those in clinical infection. The samples were then incubated at 16�C and rotated once daily. Motility and morphology, viewed using computer-assisted sperm analysis (CASA: Spermvision; Minitube of America, Verona, WI, USA), and pH (Accumet AB15, Fisher Scientific, Hanover Park, IL, USA) were measured daily for each sample at 25�C. Data from 4 replicates were used in the analysis. For motility, ANOVA revealed no significant differences (P < 0.05) between the control and inoculated samples. A PROX MIXED analysis (SAS, SAS Institute, Inc., Cary, NC, USA) revealed no treatment-by-time interaction with sperm motility after inoculation. For sample pH, statistically significant differences (P < 0.05) were noted between all of the samples, primarily contributed by a treatment-by-time effect. The pH of the control sample became more basic over the 7-day period (from 6.94 to 7.32). This phenomenon was also observed in all of samples; however, semen inoculated with AX appeared to remain closer to neutral pH than did the RP samples. Although statistically significant differences were noted in pH, the addition of biofilm bacteria did not negatively affect the motility of extended porcine semen during this time period. Further experiments need to be performed in relation to different concentrations, time period of bacterial growth, and determination of final cfu mL–1.

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12PREDICTION OF BULL FERTILITY BY COMPUTER ASSISTED SEMEN ANALYSIS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab12 ◽

2004 ◽

Vol 16 (2) ◽

pp. 128 ◽

Cited By ~ 3

Author(s):

S. Cseh ◽

T. Polichronopoulos ◽

L. Solti

Keyword(s):

Sperm Motility ◽

Semen Analysis ◽

Computer Assisted ◽

Reproductive Process ◽

Return Rates ◽

Frozen Semen ◽

Chi Square Analysis ◽

Fertilizing Capability ◽

Weighted Values

Sperm motility is clearly essential for fertilization both in vivo and in vitro. Motility is necessary for successful sperm transport, a step that is bypassed with in vitro fertilization. Recently, increasing attention has been paid to the objective evaluation and characterization of sperm motility more than simply determining the total proportion of motile spermatozoa. The purpose of computerassisted semen analysis (CASA) is to provide values for sperm concentration and sperm motility more rapidly and accurately than those obtained with traditional semen analyses methods. The objective of our experiment was to investigate the effect of specific aspects of sperm movement, such as the velocity of progression and the actual pattern of movement, to the fertilizing capability of sperm. Frozen semen samples of 10 HF breeding bulls were used in the study. For the motility analyses, Medealab CASA system (Medealab, Germany, Ver. 4.1) was used, and the velocity parameter of VCL (curvalinear velocity, μms−1), VSL (straight line velocity, μms−1), and VAP (average path velocity, μms−1) were evaluated and compared with the Day 30 and 75 non−return rates (NR30 and NR75). For every sample, a total of 10 fields were examined for 8s using a disposable 20 micron capillary chamber (CellVision, USA) giving a total of 1165 to 2831 cells evaluated. Chi square analysis, analyses of variance and linear correlation coefficient was applied to the statistical evaluation and comparison of the results. Data are based on weighted values. From the same batch of the analyzed frozen semen, a total of 8099 females were inseminated in more than 100 farms with a total of 6590 animals being positive for pregnancy at Day 30 and 4525 animals at Day 75. Within the bulls, differences were found in the values of NR30 and NR75 (P<0.05). Our data indicate very strong differences between the males’ NR30 and NR75 values (NR30: 65.6%±13.04 to 79.6%±11.17; P<0.001 and NR75: 37.8%±10.38 to 58.3%±15.53; P<0.001) reflecting the individual differences in the fertilizing capability of the males. All velocity parameters show very high correlation with strong significance both non−return rates but the best values belong to VAP (NR30 and NR75; P<0.02). Our data indicate that the bulls with lower VCL (25.51±33.04 to 79.54±58.03), VSL (11.35±19.45 to 36.36±35.71), and VAP (12.67±19.06 to 41.75±34.45) values showed lower fertilization rates both at NR30 and NR75. Computer and video technologies have advanced rapidly in recent years; thus the capability and accuracy of the latest versions of CASA systems are considerably better and they give more information about the different motion characteristics of spermatozoa. Because of the vital role of sperm motility in the reproductive process, such systems will enable us to move into a new era of diagnostic andrology and predict the fertilizing capability of semen. Supported by NKFP-Grants 4/040/2001 and 4/031/2001.

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Effect of Panax notoginseng Extracts on Inferior Sperm Motility in vitro

The American Journal of Chinese Medicine ◽

10.1142/s0192415x9900015x ◽

1999 ◽

Vol 27 (01) ◽

pp. 123-128 ◽

Cited By ~ 15

Author(s):

Jung-Chou Chen ◽

Ming-Xiong Xu ◽

Leih-Der Chen ◽

Yan-Nian Chen ◽

Tsan Hung Chiu

Keyword(s):

Sperm Motility ◽

Sperm Count ◽

Semen Analysis ◽

Panax Notoginseng ◽

World Health ◽

Computer Assisted ◽

Butanol Extract ◽

The World ◽

Health Organization

The purpose of this study was to investigate the effects of Panax notoginseng extracts on inferior sperm motility in vitro. Semen samples were collected from 23 patients with sperm motility between 20% and 40%. The sperm count was over 20 × 106/ml in accordance with the World Health Organization standard. 1.0 mg/ml and 2.0 mg/ml of Panax notoginseng extracts including aqueous extract, n-butanol extract, and polysaccharide fraction on sperm motility and progression were evaluated by computer assisted semen analysis. The results demonstrated that sperm motility as well as progression on inferior sperm motility were enhanced at 1 hour and 2 hours after incubation with all three types of extracts.

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Evaluation of stallion sperm motility with ImageJ using a cell phone camera and a light microscope

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-11934 ◽

2020 ◽

Vol 72 (6) ◽

pp. 2007-2016

Author(s):

G.P. Freitag ◽

L.G.F. Lima ◽

L.E. Kozicki ◽

L.C.S. Felicio ◽

R.R. Weiss

Keyword(s):

Sperm Motility ◽

Light Microscope ◽

Cell Phone ◽

Visual Analysis ◽

Semen Analysis ◽

Reference Method ◽

Computer Assisted ◽

Lower Accuracy ◽

Chi Squared ◽

A Cell

ABSTRACT This study aimed to determine the accuracy of assessing stallion sperm motility using a light microscope, a cell phone camera, and a free computer-assisted semen analysis (FCASA) package for ImageJ. The total motility of frozen (n=22) and cooled (n=48) equine semen was determined by FCASA and compared to the results of subjective visual analysis (SVA) by two technicians. Frozen samples were also evaluated by a commercial computer-assisted semen analysis (CCASA) system. The Friedman test revealed no significant differences (P>0.05) between cooled samples analyzed by FCASA (38.0) and SVA (technician 1, 40.0; technician 2, 40.0), nor between frozen samples analyzed by FCASA (23.36 ± 15.9), SVA (25.5 ± 18.8 and 25.8 ± 18.5), and CCASA (25.2 ± 18.3). However, mean FCASA results were underestimated by 7.2% compared with CCASA. The correlation between FCASA and CCASA was significant and strong (P<0.0001, r=0.95). Chi-squared tests indicated that FCASA provided similar results (P=0.14) to the reference method (CCASA), but SVA had lower accuracy (P=0.04). ImageJ analysis of cell phone videos captured under a light microscope can be used for estimation of stallion sperm motility with comparable accuracy to commercial systems.

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163 HYPERACTIVATION OF STALLION SPERM IN FOLLICULAR FLUID FOR IN VITRO FERTILIZATION OF EQUINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab163 ◽

2012 ◽

Vol 24 (1) ◽

pp. 193 ◽

Cited By ~ 5

Author(s):

A. Lange-Consiglio ◽

F. Cremonesi

Keyword(s):

In Vitro Fertilization ◽

Sperm Motility ◽

Follicular Fluid ◽

Zona Pellucida ◽

Semen Analysis ◽

Computer Assisted ◽

Vitro Fertilization ◽

Sperm Penetration ◽

Low Incidence

In vitro fertilization has remained elusive in the horse, as evidenced by low sperm penetration rates when IVF has been attempted with in vivo- or in vitro-matured oocytes. It is likely that the low sperm penetration rates observed in IVF studies are due to the inability to appropriately capacitate or hyperactivate, or both, stallion sperm in the laboratory. The acquisition of hyperactivated sperm motility has been observed within the oviducts of mammals at the time of fertilization and is required for zona pellucida penetration in conjunction with the acrosome reaction (AR). Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1 h of in vitro binding. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. Protein-bound progesterone, present in equine follicular fluid (FF), has been demonstrated to induce AR in stallion spermatozoa. In this context, the aims of this study were (1) to hyperactivate stallion sperm in FF and (2) to verify whether this hyperactivation supports equine IVF. Pooled FF, aspirated from the preovulatory follicles of oestrous mares, was used and its progesterone concentration was determined by immune enzymatic assay. Spermatozoa from fertile stallions selected by a swim-up procedure were pre-incubated for 6 h in capacitating medium (modifed Whittens's medium (WM) supplemented with 25 mM NaHCO3 and 7 mg mL–1 of BSA) and then incubated for 6 h at 37°C in either FF or capacitating WM. Sperm motility was assayed by computer-assisted semen analysis, rates of AR were assessed by fluorescein isothiocyanate-PNA staining and rate of apoptosis was assessed by an annexin V test. For IVF, spermatozoa were incubated at 10 × 106 sperm mL–1 in capacitating WM for 6 h and then diluted to 1 × 106 sperm mL–1 in capacitating WM with or without 10% of FF. Five mature mare oocytes were transferred into droplets (100 μL) of the sperm suspensions covered with mineral oil and then incubated for 18 h at 38.5°C in 5% CO2 in humidified air. After that, oocytes were transferred to an embryo culture medium (DMEM/F-12) for an additional 3 days. Data were analysed by ANOVA. Treatment of sperm with FF resulted in a significant (P ≤ 0.05) decrease of 3 motility variables indicative of hyperactivation: straight line velocity, straightness and linearity. The highest rate of AR (29.44%) and a lower rate of apoptosis (16.93%) were obtained after 4 h of incubation in follicular fluid. By coupling capacitating conditions with the induction of hyperactivation using follicular fluid, we have obtained reproducible percentages of 8-cell-stage embryos (18.56%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with FF did not fertilize (0%). It is concluded that mare FF does not impair sperm viability, stimulates equine sperm hyperactivation in vitro, induces the AR and supports equine IVF.

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18 SEMENRATE: THE USE OF COMPUTER-ASSISTED SEMEN ANALYSIS AND FLOW CYTOMETRY FOR OBJECTIVE BOVINE SEMEN ANALYSIS IN THE UNITED KINGDOM

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab18 ◽

2017 ◽

Vol 29 (1) ◽

pp. 116

Author(s):

M. W. Spilman ◽

K. L. Burton ◽

J. M. E. Statham

Keyword(s):

Flow Cytometry ◽

United Kingdom ◽

Semen Quality ◽

Semen Analysis ◽

Computer Assisted ◽

Motile Sperm ◽

Bovine Semen ◽

The United Kingdom ◽

Frozen Semen ◽

Viable Sperm

Routine assessment of bovine semen consists of a subjective assessment of morphology, motility and concentration. This subjective approach used during quality control at semen production centres (SPC) or investigations of poor reproductive performance in veterinary practice has been shown to be relatively inaccurate, imprecise, and operator dependent (Vincent, et al. 2012 Anim. Reprod. 9, 153–165). Assessment of frozen semen samples in a dedicated laboratory aimed to establish variations in multiple parameters associated with fertility using computer-assisted semen analysis and flow cytometry and evaluate their relationship to semen performance in the field. This has developed into a commercial service that is available to veterinarians and farmers across the United Kingdom. AI semen from 50 farms across Yorkshire, UK, that had been stored on farm was assessed for factors associated with fertility (motility, progressive motility, intact acrosome, viability, and polarised mitochondria). Data ranges and mean values for each parameter have been analysed. This analysis is ongoing as the dataset continues to expand and significance will be assessed. For frozen semen (n = 79), % viable sperm (max = 67.64, min = 0.00, mean = 43.44), % sperm with polarised mitochondria (max = 72.50, min = 0.26, mean = 38.56), % sperm with acrosome intact (max = 68.82, min = 0.06, mean = 35.29), % motile sperm (max = 66.90, min = 0.00, mean = 37.44) and % progressively motile sperm (max = 59.00, min = 0.00, mean = 26.11). 25% of the samples fell below the cut off for release of 30% motile sperm set by SPCs. For sexed AI semen (n = 9), % viable sperm (max = 66.31, min = 17.08, mean = 43.57), % polarised mitochondria (max = 26.74, min = 13.40, mean = 19.96), % intact acrosome (max = 52.62, min = 15.34, mean = 37.00), % motile (max = 38.00, min = 9.40, mean = 24.88) and % progressively motile (max = 22.80, min = 3.90, mean = 13.15). Objective semen analysis before beginning an embryo collection programme allows informed decisions to be made regarding semen choice and dosage depending on compensable v. non-compensable defects detected (Hudson et al. 2012 Dairy Herd Health 73–111; CABI Publishing). Use of semen that falls below the 30% cut off for SPCs is unlikely to perform as expected in the field (Phillips et al. 2004 Anim. Reprod. Sci. 80, 47–61). A European collaboration aims to establish correlations between semen quality parameters and fertility outcomes for UK cattle herds, providing unique data for the industry (Sellem et al. 2015 Theriogenology 84, 1447–1454.e5). These data should highlight to stakeholders in the industry how imperative optimal semen quality is and highlight the benefits to herd fertility and financial performance.

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180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab180 ◽

2016 ◽

Vol 28 (2) ◽

pp. 221

Author(s):

D. Le Bourhis ◽

S. Camugli ◽

P. Salvetti ◽

L. Schibler ◽

E. Schmitt

Keyword(s):

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Semen Analysis ◽

Membrane Integrity ◽

Computer Assisted ◽

Cleavage Rate ◽

Fluid Medium ◽

And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

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Statistical Andrology: Standard Semen Analysis and Computer-Assisted Sperm Motility Analysis

Archives of Andrology ◽

10.3109/01485019308987742 ◽

1993 ◽

Vol 30 (2) ◽

pp. 105-110 ◽

Cited By ~ 4

Author(s):

F. Culasso ◽

A. Lenzi ◽

L. Gandini ◽

F. Lombardo ◽

F. Dondero

Keyword(s):

Sperm Motility ◽

Semen Analysis ◽

Computer Assisted

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Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility

Reproduction ◽

10.1530/rep-12-0145 ◽

2012 ◽

Vol 144 (6) ◽

pp. 677-685 ◽

Cited By ~ 27

Author(s):

Francisco Jiménez-Trejo ◽

Miguel Tapia-Rodríguez ◽

Marco Cerbón ◽

Donald M Kuhn ◽

Gabriel Manjarrez-Gutiérrez ◽

...

Keyword(s):

Western Blot ◽

Enzymatic Activity ◽

Tyrosine Phosphorylation ◽

Sperm Motility ◽

Tryptophan Hydroxylase ◽

Semen Analysis ◽

Human Sperm ◽

Computer Assisted ◽

Serotoninergic System ◽

Peripheral Tissues

Serotonin (5-hydroxytryptamine; C10H12N2O (5-HT)) is produced in the CNS and in some cells of peripheral tissues. In the mammalian male reproductive system, both 5-HT and tryptophan hydroxylase (TPH) have been described in Leydig cells of the testis and in principal cells of the caput epididymis. In capacitated hamster sperm, it has been shown that 5-HT promotes the acrosomal reaction. The aim of this work was to explore the existence of components of the serotoninergic system and their relevance in human sperm physiology. We used both immunocytochemistry and western blot to detect serotoninergic markers such as 5-HT, TPH1, MAOA, 5-HT1B, 5-HT3, and 5HTT; HPLC for TPH enzymatic activity; Computer Assisted Semen Analysis assays to measure sperm motility parameters and pharmacological approaches to show the effect of 5-HT in sperm motility and tyrosine phosphorylation was assessed by western blot. We found the presence of serotoninergic markers (5-HT, TPH1, MAOA, 5-HT1B, 5-HT2A, 5-HT3, 5-HTT, and TPH enzymatic activity) in human sperm. In addition, we observed a significant increase in tyrosine phosphorylation and changes in sperm motility after 5-HT treatment. In conclusion, our data demonstrate the existence of components of a serotoninergic system in human sperm and support the notion for a functional role of 5-HT in mammalian sperm physiology, which can be modulated pharmacologically.

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