An osmotic resistance test for bovine semen

The objective of the study was to compare two different flow cytometers to reveal if there are differences between them and to find the most suitable protocol for analysis of spermatozoa. These two flow cytometers; Cell Lab Quanta TM and Coulter Epics XL, have different principles to calculate cell size, electric volume, and forward scatter (FS), respectively. Flow cytometry is a valuable tool to assess various spermatozoa quality traits simultaneously, such as plasma membrane and acrosome integrity. A double-and triple-stain combination was performed to compare evaluation of these two parameters by both flow cytometers and to assess the need of a fluorescent probe to identify the spermatozoa. Propidium iodide was used to assess the proportion of dead spermatozoa, whereas Alexa Fluor V R 488 conjugated peanut agglutinin (PNA-Alexa 488) was used to evaluate the percentage of acrosome intact and acrosome-reacted cells or degenerated cells. In the triple-stain protocol, MitoTracker V R Orange (MO) was included to test the capacity of this probe to discriminate spermatozoa from egg yolk and debris particles present in the semen sample. Cryopreserved semen from 13 Norwegian Red bulls was included in the study and the semen was evaluated immediately after thawing and after 3 hr incubation at 37 C. The results show that there is good agreement between the instruments. Nevertheless, a significant difference was found in percentages of acrosome intact live spermatozoa (% AIL) when including MO as a spermatozoa identification probe, compared to assessment without MO, with the Coulter Epics XL, while no significant difference was found when including the probe with the Cell Lab Quanta. In conclusion, the results show that cell size measurement based on electronic volume used by the Cell Lab Quanta flow cytometer is more accurate than FS used by the Coulter Epics XL flow cytometer in identification of spermatozoa. V C 2014 International Society for Advancement of Cytometry Key terms flow cytometry; sperm analysis; electronic volume; forward scatter; plasma membrane integrity; acrosome integrity FLOW cytometry has become a powerful tool for characterization of cell populations. The instrument has the capability to measure multiple parameters of individual cells, thus it is possible to detect abnormal cells in a short time (1-3). The flow cytometric analysis is based on light-spreading and fluorescence signals. Light that is scattered at high angles is termed side scatter (SS) and will provide information about such intracellular properties as cell granularity (4). Scattered light with a small angle is termed forward scatter (FS) and depends on particle size and the refractive

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“…The ANOVA statistics showed that bull and straw were significant sources of variation (Table 1) in accordance to published data (19,20).…”

Section: Original Articlesupporting

confidence: 86%

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa

Standerholen

Myromslien²,

Kommisrud³

et al. 2014

Cytometry Pt A

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“…An in vitro assay for determining bull fertility, which could replace ®eld fertility trials, would be of great bene®t to AI programmes. Many methods have been used for the assessment of bull sperm viability following cryopreservation including examination of motility (Kjaestad et al, 1993;Correa et al, 1997), ATP measurements (Janskauskas and Rodriguez-Martinez, 1995), membrane integrity (Harrison and Vickers, 1990;Janskauskas and Rodriguez-Martinez, 1995) and measurement of osmotic resistance (Correa and Zavos, 1994;Revell and Mrode, 1994;Rota et al, 2000). The results of such assays do not always correlate with the fertility of a semen sample.…”

Section: Introductionmentioning

confidence: 99%

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

Ward

Rizos

Corridan

et al. 2001

Molecular Reproduction Devel

128

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The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57±78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r 0.689, P`0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility. Mol. Reprod. Dev. 60: 47±55,

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“…A total of 200 spermatozoa was counted (magnification x 400) under a phase-contrast microscope. Spermatozoa with swollen or coiled tails were recorded (22).…”

Section: Semen Extending Freezing and Thawingmentioning

confidence: 99%

In vitro evaluation of Saanen buck semen frozen in different extenders supplemented with various antioxidants

Recai;DAŞKIN¹

2010

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Summary:The aim of this present study was to investigate the effects of extenders containing different antioxidants upon the principle sperm characteristics of Saanen buck semen following freezing. In this study, four bucks were used. Semen was collected twice a week by an artificial vagina during the breeding season to obtain ten ejaculates per animal. The samples were then extended in three extenders (tris, T-, skimmed milk, M-and laiciphos 488, L-based) without (as controls) or with the two antioxidants (5 mM cysteine, C and 1000 µg/ml hyaluronic acid, H). By this way, nine experimental groups were assigned, as follows:For the T extender groups, the rates of motility and viability were significantly (p<0.01) higher for the T-C extender as compared to those of the T-H and T-O. However, there was no such difference (p>0.05) among the extenders based on the rates of abnormal spermatozoa, abnormal acrosome and hypoosmotic swelling (HOS) test. For the M groups, the motility was significantly (p<0.01) higher for the M-C and M-O extenders as compared to that of the M-H. However, there was no such difference (p>0.05) among the extenders based on the rates of viability, abnormal spermatozoa, abnormal acrosome and HOS test. For the L groups, the rates of motility and viability were significantly (p<0.01) higher for the L-C extender as compared to that of the L-H but not that of the L-O. Additionally, there was no such difference (p>0.05) among the extenders mentioned above based on the rates of abnormal spermatozoa, abnormal acrosome and HOS test. As a result, the present in vitro findings of semen quality parameters studied suggest that; i) both skimmed milk and laiciphos extenders were more favourable than tris, and that ii) the cysteine, as an antioxidant, provided a superior protection than the hyaluronic acid did.

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An osmotic resistance test for bovine semen

Cited by 344 publications

References 26 publications

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

In vitro evaluation of Saanen buck semen frozen in different extenders supplemented with various antioxidants

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