303 FERTILIZATION-RELATED PARAMETERS OF FROZEN - THAWED SPERMATOZOA FROM SUBFERTILE JAPANESE BLACK CATTLE

2007 ◽  
Vol 19 (1) ◽  
pp. 266
Author(s):  
K. Kuroda ◽  
M. Fukushima ◽  
M. Miyake ◽  
H. Harayama
Keyword(s):  
Acrosome Reaction ◽  
Economic Losses ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Causal Factors ◽  
Mean Values ◽  
Progressive Movement ◽  
Male Subfertility ◽  
Fertilizing Ability

The subfertility derived from male factors is a problem of concern in domestic animals, because it could cause a disintegration of the breeding system and large economic losses, particularly when the subfertility affects genetically superior male animals. Therefore, it is urgent that causal factors of male subfertility be determined. Recently, an increasing number of subfertile bulls have been found among Japanese Black cattle, which is a representative breed of Japanese beef cattle. The purpose of the present study was to elucidate causal factors of male subfertility in Japanese Black cattle. Frozen–thawed spermatozoa from 8 subfertile (S1-S8) and 7 fertile (F1–F7, control) bulls were used for the assessment of fertilization-related parameters. The data obtained from each subfertile bull in the following experiments were individually compared with the mean values of the fertile bull group. In Experiment 1, sperm motility was observed in samples that were frozen-thawed and subsequently washed in PBS. Many spermatozoa (higher than 65%) exhibited flagellar movement in all samples from fertile and subfertile bulls. However, the percentages of progressively motile spermatozoa from 2 subfertile bulls were significantly lower (S2: 6%; S7: 7%; P < 0.05, ANOVA and Tukey's multiple range tests) than those from fertile bulls (average: 37%). Moreover, rapidly progressive movement was not observed in the spermatozoa from 4 subfertile bulls (S1, S2, S6, and S7). These data suggest abnormality in the motility system of sperm flagella in these 4 subfertile bulls. In Experiment 2, the capacitation–acrosome reaction state of frozen–thawed spermatozoa was examined by the CTC-staining assay. More than 50% of the frozen–thawed spermatozoa from 4 subfertile bulls (S5–S8) were prematurely progressing in the capacitation state immediately after washing and resuspension in the medium lacking CaCl2. Moreover, the addition of CaCl2 to the medium induced acrosomal loss in these sperm samples (percentages of spermatozoa without the acrosome: 36–49%). These findings indicate the occurrence of premature capacitation and a spontaneous acrosome reaction in spermatozoa from these 4 subfertile bulls. In Experiment 3, the in vitro fertilizing ability of frozen–thawed spermatozoa was evaluated by the IVF test. The percentages of fertilized eggs with both male and female pronuclei or developmental rates of fertilized eggs to the 2-cell or 4-cell stage were significantly lower in the spermatozoa from S6 to S8 bulls than in those from fertile bulls (P < 0.05, chi-squared tests). This may suggest that spermatozoa from these 3 subfertile bulls hardly accomplish the normal fertilization process. In summary, low progressive motility and low in vitro fertilizing ability because of premature capacitation were found in the spermatozoa from subfertile bulls. It is therefore possible that these are causal factors for the subfertility of male Japanese Black cattle.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 93-99 ◽  
Author(s):  
Hiroyuki Tateno ◽  
Yujiroh Kamiguchi
Keyword(s):  
Gas Phase ◽  
Acrosome Reaction ◽  
Chinese Hamster ◽  
Subsequent Development ◽  
In Vitro Fertilisation ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Potential Use

SummaryTo enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2 in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2 in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 152
Author(s):  
M. A. Lagares ◽  
H. S. Martins ◽  
M. R. Souza ◽  
C. F. A. M. Penna ◽  
F. O. P. Leme ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Propidium Iodide ◽  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Peanut Agglutinin ◽  
Binding Assays ◽  
Frozen Semen ◽  
Fertilizing Ability

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P < 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P > 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P < 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P < 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen. Acknowledgments are extended to CAPES, Brazil, for the financial support.

Influence of progesterone on boar sperm capacitation

1995 ◽  
Vol 144 (1) ◽  
pp. 13-18 ◽  
Author(s):  
B Barboni ◽  
M Mattioli ◽  
E Seren
Keyword(s):  
Pisum Sativum ◽  
Direct Effect ◽  
Acrosome Reaction ◽  
Sperm Capacitation ◽  
Fertilizing Ability ◽  
Boar Sperm ◽  
The Status ◽  
Boar Semen ◽  
Fertilization System

Abstract This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITCconjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation. Journal of Endocrinology (1995) 144, 13–18

154 CRYO-ACROSOME REACTION/CAPACITATION OF EJACULATED AND EPIDIDYMAL BOVINE SPERM AND THEIR IN VITRO FERTILIZATION RATES

2012 ◽  
Vol 24 (1) ◽  
pp. 189
Author(s):  
M. A. Stout ◽  
J. R. Saenz ◽  
J. F. Chenevert ◽  
G. T. Gentry ◽  
K. B. Bondioli ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Fertilization Rate ◽  
Fluorescent Staining ◽  
Epididymal Sperm ◽  
Vitro Fertilization ◽  
Mean Values ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
The Mean

Seminal plasma has been shown to affect the composition and function of sperm. This exposure may alter the ability of sperm to endure cryopreservation, undergo capacitation and fertilize oocytes. Earlier studies demonstrated that the freezing response of epididymal and ejaculated sperm from the bulls utilised in these studies was similar (Alapati et al. 2009). Subsequent studies are designed to investigate the response of ejaculated and epididymal bovine sperm to cryopreservation and their ability to undergo capacitation. Ejaculated and epididymal sperm from the same bulls (n = 4) were collected by an artificial vagina and retrograde caudal epididymal flush, respectively and were cryopreserved in standard egg-yolk glycerol extender. Sperm was compared by level of cryo-acrosome reaction/capacitation and their fertilization rates in vitro with and without the capacitating agent heparin. Upon evaluation, a sample was thawed and washed and the viable sperm population was isolated through a discontinuous Percoll® gradient. Sperm viability and acrosome status were assessed by fluorescent staining with propidium iodine and fluorescein isothiocyanate-PNA followed by evaluation with flow cytometry. Capacitated sperm were then induced to undergo the acrosome reaction by exposure to lysophosphatidylcholine (10 μg mL–1). Cryo-capacitation was determined by the difference between cryo- and lysophosphatidylcholine-induced acrosome-reacted sperm levels. Differences in the mean values between groups were analysed by ANOVA. Ejaculated and epididymal sperm differed by level of cryo-acrosome reaction (15 vs 4%, respectively; P < 0.001), but did not differ by level of cryo-capacitation (6.2 vs 5.9%, respectively; P = 0.92). The ability of epididymal and ejaculated sperm to fertilize oocytes in vitro with and without heparin was also investigated. Sperm were thawed and washed and the viable population was isolated through a Percoll® gradient. Oocytes were washed and randomly assigned to a treatment group, either ejaculated ± heparin or epididymal ± heparin. Oocytes and sperm were added to a capacitating medium (TALP) with and without heparin. Following 18 h of incubation, fertilization rate was determined by pronuclear fluorescent staining with Hoechst 33342 followed by confirmation with aceto-orcein staining. Differences in the mean values among treatment groups were analysed by one-way ANOVA followed by Holm-Sidak pairwise multiple comparisons. Fertilization rates of epididymal sperm with and without heparin and ejaculated sperm with heparin were similar (76, 80 and 70%, respectively). Ejaculated sperm without heparin was lower than all other groups, with a fertilization rate of 42% (P < 0.001). In summary, epididymal sperm displayed lower levels of cryo-acrosome reaction but were similar by level of cryo-capacitation. In addition, epididymal and ejaculated sperm differed in the need for a capacitating agent such as heparin when used in vitro. This may be useful to increase efficiency when using epididymal sperm in assisted reproductive techniques.

Gene expression profile differences in embryos derived from prepubertal and adult Japanese Black cattle during in vitro development

2012 ◽  
Vol 24 (2) ◽  
pp. 370 ◽  
Author(s):  
Dorji ◽  
Yukihiro Ohkubo ◽  
Kazuchika Miyoshi ◽  
Mitsutoshi Yoshida
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Bovine Genome ◽  
Gene Expression Profiles ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24 072 probe sets representing over 23 000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24–35 months old) versus prepubertal (9–10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.4%) and 490 (2.0%) genes were differentially expressed in prepubertal and adult bovine in 8- to 16-cell and blastocyst stage embryos, respectively. Out of these, 218 and 248 genes were upregulated, while 373 and 242 were downregulated in prepubertal and adult 8- to 16-cell and blastocysts stage embryos, respectively. Gene ontology classification regarding biological process, molecular functions and cellular component revealed diversity in transcript abundances between prepubertal and adult groups in both the distinct developmental stages. Quantitative reverse transcription–PCR validated the expression differences of some selected transcripts as identified by microarray analysis. To our knowledge, this is the first report indicating the significant number of genes differentially expression (>2-fold, P < 0.01) in preimplantition embryos between adult and prepubertal Japanese Black cattle during in vitro development.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 165-171
Author(s):  
M. Kishi ◽  
R. Takakura ◽  
Y. Nagao ◽  
K. Saeki ◽  
Y. Takahashi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Cell Cycle Stage

SummaryIn the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

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