Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro (original) (raw)
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Sperm Penetration Assay as an Indicator of Bull Fertility
Journal of Reproduction and Development, 2012
To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cutoff value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.
In vitro assessment of sperm from bulls of high and low field fertility
Theriogenology, 2011
The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 o C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 o C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilise oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalise residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95%CI 39.5 to 75.3) compared to the low fertility 3 (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95%CI 60.9 to 89.4) and 55.3 (95%CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95%CI 21.1 to 49.6) and 24.2 % (95%CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilise oocytes in vitro; however, once fertilisation occurred subsequent embryo development was not significantly affected by fertility status.
2015
The ability to penetrate zona-intact oocyte has been a very useful test of sperm fertility. In this study, the accuracy of bioassay in predicting sperm fertility using water buffalo oocytes matured in vitro have been evaluated. In Experiment 1, length of sperm-oocyte coincubation was evaluated based on percentage penetration. The observed mean penetration rate using 10 hr and 24 hr co-incubation time had no difference, including monospermic fertilization rate. In Experiment 2, the effect of caffeine, theophylline alone or in combination were evaluated based on percentage penetration rate. Neither conditions with capacitating agents used showed no significant differences in the mean penetration rates ( but was higher significantly against the control (P<0.05). In Experiment 3, 5 bulls were evaluated on their fertilizing capability. The mean penetration rates observed among the bulls evaluated had no significant differences (34.44 ± 8.68 to 48.61 ± 2.32). The results of this study ...
Development of in vitro tests to predict fertility of bulls
Canadian Journal of Animal Science, 2005
of in vitro tests to predict fertility of bulls. Can. J. Anim. Sci. 85: 47-52. The overall objective was to develop an in vitro test to predict fertility of bulls in the field. We investigated the bull effect on in vitro embryo production, zona binding and acrosome reaction, and the correlation of this effect to field fertility measured by 60-90 d non-return rate. Frozen semen from three separate ejaculates of eight unrelated young bulls, obtained from an artificial insemination (AI) center, was used. On thawing, ejaculates from each bull were pooled, motile sperm were selected and (a) subjected to immunofluorescent assay at 0 and 4 h of incubation in capacitation medium to assess acrosome status, (b) used in an in vitro fertilization assay system to assess cleavage and blastocyst production rates, and (c) sperm-zona binding assay was carried out to determine the number of sperm bound to the zona pellucida of mature oocytes. Percentage of pre-freeze motile sperm (PrFM) and non-return rate data were obtained from the AI center. PrFM, percentage of acrosome reacted sperm at 0 h (AR1), increase in percentage of acrosome reacted sperm after 4 h (InAR) and sperm-zona binding rates (ZB) differed (P < 0.05) among sperm samples obtained from different young bulls. Significant correlations (P < 0.05) were observed between PrFM and AR1 (r = -0.31), InAR (r = 0.36), and ZB (r = 0.32). AR1 was negatively correlated to ZB (r = -0.27) and cleavage rate (r = -0.20), InAR was positively correlated with ZB (r = 0.31) and cleavage rate (r = 0.26). None of the in vitro tests was correlated with non-return rate. These findings indicate that along with pre-freeze motility, a combination of in vitro tests including the percentage of spontaneously acrosome reacted sperm at thawing, might be useful in predicting bull field fertility. Such a combination of assays, however, has yet to be determined.
Theriogenology, 2007
In the present study, we aimed to develop a polyacrylamide gel that could be used instead of bovine cervical mucus in the cervical mucus penetration test (CMPT) to obtain coherent and replicable results in bulls. The frozen semen samples of six Holstein bulls, which were divided into two fertility groups as low and high according to their non-return rate (NRR), were used. In this study, the modified CMPT (mCMPT) was carried out within 0.25 mL transparent plastic straws with an inner diameter 1.7 mm. The penetration ability of spermatozoa to bovine cervical mucus and to polyacrylamide gels swollen with two different solutions [NaCl (G1) and PBS (G2)] was compared. For the penetration test, the straws filled with cervical mucus and both gels were dipped into thawed semen samples and incubated at 37 8C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at À20 8C. On the evaluation day, the frozen straws were cut at 1.5-1.75 cm (penetration distance range = PDR1), 3.25-3.5 cm (PDR2) and 5.0-5.25 cm (PDR3), beginning from open-end of the straws. The separated frozen parts were then immediately transferred onto special counting slides by pushing with a mandrel and left to thaw. Thawed samples were covered with cover glass and penetrated spermatozoa in these parts were counted. The relation between the results and fertility of bulls was determined.
Evaluation of bull semen for fertility-associated protein, in vitro characters and fertility
Proteins present in the seminal plasma and sperm influence sperm function and fertilization. The present study was carried out to screen breeding bull semen samples for the presence of fertilityassociated 28–30 kDa heparin-binding protein (HPB) and its effect on in vitro sperm characters and fertility. Semen samples were collected from 22 breeding bulls and the sperm proteins were extracted by Triton X detergent extraction method. HBPs were eluted, and the molecular weight of the proteins was assessed by discontinuous sodium dodecyl sulphate polyacrylamide gel elecrophoresis. Based on the presence/absence 28 kDa HBPs, bulls were categorized into group I and group II. Frozen semen samples were evaluated for in vitro sperm characters at immediate post thaw, 60, 120 and 180 min post-thaw incubation. To assess the field fertility of the bulls, 50 frozen semen straws/bull were used for insemination. Results indicated that only 50% of the bulls screened had 28–30 kDa HBPs in their sperm. Bulls positive for fertility-associated protein had better in vitro sperm characters, better protection against oxidative stress, readily underwent capacitation induction by heparin and had 13% higher conception than the bulls lacking the protein. So, it can be concluded that the bulls positive for of 28–30 kDa HBPs in sperm had higher chance of fertility and screening for its presence can be included in the regular breeding soundness examination for selection of bulls.
Italian Journal of …, 2010
The aim of this work was to evaluate the effect of bull on the efficacy of different The aim of this work was to evaluate the effect of bull on the efficacy of different capacitating agents in buffalo. Spermatozoa derived from 4 different bulls were incubated in absence tozoa derived from 4 different bulls were incubated in absence of a capacitating agent, in presence of 0.01 mM heparin and in 20% buffalo estrous serum (BES) for 2 0.01 mM heparin and in 20% buffalo estrous serum (BES) for 2 hours. Sperm were then e�posed to lysophosphatidylcholine, an agent that induces acrosome reaction Sperm were then e�posed to lysophosphatidylcholine, an agent that induces acrosome reaction only on capacitated spermatozoa. A double staining technique with Trypan-blue/Giemsa was used to A double staining technique with Trypan-blue/Giemsa was used to was used to evaluate viability and acrosome status of spermatozoa fi�ed in �7% formaldehyde. The efficiency of The efficiency of capacitation was evaluated by assessing the percentage of acrosome-reacted (AR) spermatozoa in the treated groups. A bull effect on sperm capacitation in vitro was demonstrated, as indicated by differences in the percentage of AR sperm among bulls regardless of the treatment. In particular when heparin was used as capacitating agent bull C gave higher percentages of AR sperm than bull A (22.7 vs. 14.0%, respectively) with intermediate values for bulls B and D (1��.7 and 1��.1%, respectively), whereas when BES was used bull D was the most efficient one (22.1, 21.��, 2�.0, and ��.0% for bulls A, B, C and D, respectively).
Livestock Science, 2009
This study aimed to develop a system of in vitro assays based on zona pellucida binding and in vitro fertilization for predicting male fertility in buffalo bulls. Frozen-thawed semen from nine bulls was tested for motility, viability index, acrosomal integrity, zona pellucida binding and in vitro fertilizing ability. Differences in post-thaw sperm motility between bulls were not significant. Differences in viability indices and percentage of spermatozoa with detached acrosome between bulls was highly significant (P b 0.001). Sperm attached per ovum, fertilization rates and polyspermy percentages varied significantly (P b 0.01) among buffalo bulls. A significant (P b 0.01) positive correlation coefficient of 0.69 was evident between normal acrosome and sperm attached per ovum, while between normal acrosome and fertilization efficiency it was 0.72. Sperm from different buffalo bulls differs in their ability to bind and fertilize oocytes. This study provides a basis to predict and maximize the in vitro fertilization performance of individual bulls.
Bull Fertility and Its Relation with Density Gradient Selected Sperm
Volume 11, Number 1, Apr-Jun 2017
Background: Sperm selection method is usually used to collect these cells for in vitro-assisted reproduction. Few studies reported the relationship of in vivo fertility and semen parameters after sperm selection; hence, the present study attempted to assess different semen parameters after post-thaw or sperm selection, using density gradient separation BoviPure®, to predict in vivo fertility.