Methodological factors affecting the results of staining frozen–thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status

Almadaly, Essam A.; El-Kon, Ismail; Heleil, B.; Fattouh, El-Sayed; Mukoujima, Koushi; Ueda, Takuya; Hoshino, Youichirou; Takasu, Masaki; Murase, Tetsuma

doi:10.1016/j.anireprosci.2012.10.016

Cited by 18 publications

(23 citation statements)

References 38 publications

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“…Previous studies reported that ConA, PNA and PSA bind specifically to the acrosome region of bovine sperm [ 21 , 22 ]. It has been reported that the sperm acrosome may be damaged by smearing onto a glass slide, so that the glycocalyx in the acrosome region may have been damaged in a few sperm in our experiments [ 33 ]. Possibly, sperm with intact acrosomes showed pattern P1 and a few with damaged acrosomes showed other patterns with ConA, PNA and PSA labeling.…”

Section: Discussionmentioning

confidence: 99%

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation

Umezu

Hiradate

Numabe

et al. 2017

Journal of Reproduction and Development

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Sperm sorting by flow cytometry is a useful technology in the bovine industry, but the conception rates after artificial insemination using sex-sorted sperm are lower than when using the un-sorted sperm. In this study, we have investigated the causes for these low conception rates. We have focused on changes caused by flow cytometry to the glycocalyx, which forms the outermost surface of the sperm membrane. We have also evaluated the effects of capacitation on the glycocalyx since capacitation involves a redistribution of the sperm membrane that is vital for successful fertilization and conception. Lectin histochemistry was used to visualize the structure of the sperm glycocalyx. Lectin-staining sites were examined in non-treated sperm, sex-sorted sperm, and capacitated sperm. We have detected six different staining patterns related to different labeling regions of the sperm. Phaseolus vulgaris–erythroagglutinin (PHA-E) lectin-staining patterns of non-treated sperm were very different from those observed for sex-sorted sperm or capacitated sperm, suggesting that both, sex sorting by flow cytometry and the capacitation process affected the glycocalyx structures in the sperm. In addition, the total tyrosine-phosphorylation level in sex-sorted sperm was significantly higher than that in the non-treated sperm. Therefore, we concluded that the unexpected capacitation of bovine sperm during flow cytometry is associated with changes in the glycocalyx. Since premature capacitation leads to low conception rates, this unexpected capacitation could be a cause of low conception rates after artificial insemination using sex-sorted sperm.

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Section: Discussionmentioning

confidence: 99%

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation

Umezu

Hiradate

Numabe

et al. 2017

Journal of Reproduction and Development

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“…The acrosome can be observed using phase contrast microscopy [1], the triple stain technique [22], or Coomassie blue labeling [8]. More recently, fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA), which specifically binds to the sugar Galactosyl β-1,3 N-acetylgalactosamine in acrosomal membranes [13], has been used as a probe to visualize acrosomal integrity by Kishida et al .…”

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confidence: 99%

“…In our previous report on staining with FITC-PNA, spermatozoa were fixed with 4% paraformaldehyde (PFA) for 30 min, permeabilized with 1% Triton X-100 for 5 min, stained with FITC-PNA, and mounted in 1,4-diazabicyclo [2,2,2] octane (DABCO) for analysis according to our own criteria for categorization [1]. When frozen-thawed Japanese Black bull spermatozoa were stained with FITC-PSA, the labeling was not confined to the acrosomal region and there was non-specific labeling of the head and flagellum, whereas FITC-PNA labeling was specific to the acrosomal region [1]. Therefore, FITC-PNA is more suitable for assessing acrosomal status in bull spermatozoa.…”

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confidence: 99%

“…Since PNA binds to molecules present inside the sperm plasma membrane, it is necessary to permeabilize spermatozoa before they are stained with PNA to reveal the integrity of the acrosome. For frozen-thawed bull spermatozoa, treatment with 1% Triton X-100 for 5 min has been used to permeabilize acrosomes for staining with FITC-PNA [1, 5]. However, because Triton X-100 is also used to extract proteins from cells [3, 6, 17], reducing its concentration might minimize the acrosomal damage caused by excessive treatment.…”

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confidence: 99%

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Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa

et al. 2019

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This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1–3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0–2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

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“…A number of studies have focused on the evaluation of acrosome status (Kitiyanant et al 2002;Puente et al 2011;Almadaly 2012), sperm capacitation and variations among different bull breeds (Demyda-Peyras et al 2012). There are also numerous studies concerned with the high variability in embryo production under in vitro conditions, using spermatozoa from different bulls, but only a few studies have investigated a potential relationship between the in vitro-induced acrosome reaction and in vivo fertility of bulls (Whitfield and doi: 10.17221/8437-VETMED Parkinson 1995;Januskauskas et al 2000;Birck et al 2010;Kumar et al 2014).…”

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Relationship between acrosome integrity changes and in vitro fertilising ability of bovine spermatozoa

Rečková¹,

Machatkova²,

Máchal³

et al. 2015

Vet. Med.

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This study was designed to investigate the characteristics of acrosomal changes during capacitation of bovine spermatozoa in relationship to in vitro fertility of individual bulls. Motile spermatozoa were separated from frozen-thawed semen by a swim-up procedure and capacitated in IVF-TALP medium with or without heparin. The spermatozoa were evaluated in terms of acrosomal changes at 0, 3, 4, 5, 6 and 8 h of capacitation. Proportions of acrosome-reacted spermatozoa at 5 h and 0 h of capacitation were used for calculation of the heparin response index. Variations in the heparin response index were found among individual bulls. Based on the mean response index value of all bulls, they fell into three categories: bulls with greater, intermediate and no response to heparin (GRH, IRH and NRH, respectively). Differences in the heparin response index between the bull categories were significant (P < 0.05). Higher D7 and D8 embryo development rates were found in the IRH vs. NRH bulls (P < 0.05). In conclusion, this study shows that the spermatozoa of bulls with a greater or intermediate response to heparin appear to be most suitable for in vitro embryo production compared with spermatozoa of bulls with no response to heparin.

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Methodological factors affecting the results of staining frozen–thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status

Cited by 18 publications

References 38 publications

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa

Relationship between acrosome integrity changes and in vitro fertilising ability of bovine spermatozoa

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