The importance of trace minerals copper, manganese, selenium and zinc in bovine sperm–zona pellucida binding

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 89-96 ◽  
Author(s):  
Juan Patricio Anchordoquy ◽  
Juan Mateo Anchordoquy ◽  
Raúl Martín Lizarraga ◽  
Noelia Nikoloff ◽  
Ana Malen Pascua ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Membrane Integrity ◽  
Trace Minerals ◽  
Bovine Sperm ◽  
Copper Manganese ◽  
Total Antioxidant ◽  
Functional Membrane ◽  
Bovine Spermatozoa ◽  
The Mean

SummarySperm–zona pellucida (ZP) binding is a necessary event for successful fertilization. The aim of this study was to determine the effect of trace minerals such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) on bovine spermatozoa binding to ZP. Sperm viability, functional membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation (LPO) were also evaluated. For the present study, in vitro fertilization (IVF) medium was supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), all minerals (Cu+Mn+Se+Zn), or tested without supplement (Control). Considerably more sperm bound to ZP when Cu, Se or Zn were added to the IVF medium, but there were no difference compared with the Control, Mn and Cu+Mn+Se+Zn groups. After 1 h of incubation, viability was increased by the addition of Cu, Mn and Se with respect to the Control but, after 2 h, viability was higher only with the addition of Mn to IVF medium. Functional membrane integrity improved in sperm treated with Cu. Acrosome integrity was higher in sperm treated with Zn after 1 h of incubation. LPO was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn. The mean TACs of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were lower than in the Control. In conclusion, the results obtained in the present study determined that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to the ZP, highlighting the importance of these minerals in the fertilization process.

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Osamu Okitsu ◽  
Shuji Yamano ◽  
Toshihiro Aono
Keyword(s):  
Human Sperm ◽  
Human Spermatozoa ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Bovine Sperm ◽  
Female Pronucleus ◽  
Sperm Factor ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.

Evaluation of chromatin integrity of motile bovine spermatozoa capacitated in vitro

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Z. Reckova ◽  
M. Machatkova ◽  
R. Rybar ◽  
J. Horakova ◽  
P. Hulinska ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Production Efficiency ◽  
Sperm Chromatin ◽  
Percoll Gradient ◽  
Embryo Production ◽  
Before And After ◽  
Bovine Spermatozoa ◽  
Chromatin Integrity ◽  
The Mean

SummaryThe efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF–TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n = 3, n = 5 and n = 9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p ≤ 0.01), as compared with the value before separation. Capacitacion produced a significant decrease in the mean non-DFI-sperm proportion in H+ sperm (p ≤ 0.01). In DNA-s bulls, separation significantly increased the mean non-DFI-sperm proportion (p ≤ 0.01) but during capacitacion, the mean non-DFI-sperm proportion remained almost unchanged. In DNA-ms bulls, neither separation nor capacitacion had any effect on the mean non-DFI-sperm proportion. It can be concluded that, although separation and capacitacion may produce some changes in sperm chromatin integrity, these are not associated with different in vitro fertility of the bulls involved.

227 CALRETICULIN, A 60-kDa PROTEIN, PREVENTS POLYSPERMY IN ZONA PELLUCIDA-FREE PIG OCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 261
Author(s):  
R. Romar ◽  
C. Soriano-Úbeda ◽  
M. D. Saavedra ◽  
J. Gadea ◽  
M. Avilés ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Calcium Binding ◽  
Statistical Significance ◽  
P Value ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Chaperone Protein ◽  
The Mean

After gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. Calreticulin (CRT), a calcium-binding highly conserved protein of 60 kDa, is contained in cortical granules from hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60), and we recently showed it is exocytosed from chemically activated ZP-free pig oocytes (Romar et al. 2012 Reprod. Fertil. Dev. 24). When pig ZP-enclosed oocytes were incubated with CRT, monospermy was not improved (Romar et al. 2011, Maternal communication with gametes and embryo, p. 72), suggesting that the likely role of CRT in preventing polyspermy might be carried out at the oolemma level. Our objective was to evaluate whether CRT prevents polyspermy in pig ZP-free oocytes by treating the cells with this protein before being inseminated. In vitro-matured cumulus–oocyte complexes (44 h, NCSU-37 medium) were decumulated and ZP was digested with Tyrode’s acid. The ZP-free oocytes were incubated for 30 min in TALP medium supplemented with 0, 100, 1000, and 5000 pg of CRT (ab91577, Abcam, Cambridge, MA, USA) per oocyte. After washing, ZP-free oocytes were inseminated (25 000 sperm mL–1) and gametes were co-cultured for 18 h. Putative zygotes were fixed and stained with Hoechst 33342 to analyse the fertilization results. Four replicates with 30 to 35 oocytes per group were done, and results were analysed by one-way ANOVA. A P-value ≤0.05 was taken to denote statistical significance. Incubation with CRT did not affect penetration rates that were similar among groups (77.12 ± 3.88 and 72.73 ± 4.07, respectively, for the 0- and 5000-pg CRT groups). However, the mean number of sperm per penetrated oocyte decreased from 3.01 ± 0.28 (0-pg group) to 2.07 ± 0.16 (5000-pg group), and monospermy rate increased from 30.77 ± 4.87 (0-pg group) to 52.27 ± 5.36 (5000-pg group; P ≤ 0.05). Incubation with CRT did not affect the number of sperm attached to oolemma, which was similar among all groups (11.45 ± 1.16 v. 10.75 ± 1.17, respectively, for 0 and 5000 pg of CRT). These preliminary data suggest that CRT, a protein exocytosed after oocyte activation, participates in the membrane block to polyspermy in pigs. Future studies to describe the exact mechanism of action of this chaperone protein are necessary. Supported by MEC and FEDER (AGL2009-12512-C02-01).

Human oocyte cytometry and fertilisation rate after subzonal insemination

Zygote ◽  
1995 ◽  
Vol 3 (2) ◽  
pp. 101-109 ◽  
Author(s):  
Jean Philippe Wolf ◽  
Sylvie Bulwa ◽  
Daniel Rodrigues ◽  
Pierre Jouannet
Keyword(s):  
Zona Pellucida ◽  
Human Oocyte ◽  
Oocyte Diameter ◽  
Antisperm Antibodies ◽  
Subzonal Insemination ◽  
Fertilisation Rate ◽  
Defect Groups ◽  
The Mean ◽  
Dynein Arms

SummaryThe cytometry of 545 oocytes was evaluated during subzonal insemination (SUZI; 85 attempts), on day 0 (egg retrieval and SUZI), day 1 and day 2(embryo transfer). On day 0, the egg and oolemma diameters (mean ± SD) were 164.0 ± 19.6 μm and 114.2±16.8 μ5m respectively.The zona thickness was 17.8± 13.4 μm and correlated with the oolemma diameter(r = 0.24, p < 0.001). The fertilisation rate was significantly lower for the smaller oocytes (less than 108 μm diameter) compared with the larger oocytes (over 108μm) (9.8% vs 21.2% respectively; p < 0.05). These was little variation in oocyte diameter according to nuclear status. However, oocyte diameter increased significantly between day 0 and day 1 (p < 0.001) for both fertilised and unfertilised oocytes. Six different indications for SUZI were investigated in detail: three with non-specific (normal and subnormal sperm with in vitro fertilization failure, oligoasthenospermia) and three with specific sperm defects (flagellar dyskinesia, absence of outer dynein arms, antisperm antibodies). Oocytes from the non-specific defect groups had significantly smaller diameters than the others (p < 0.05). The mean fertilisation rate was related to the mean oolemma diameter for the groups with non-specific sperm defects and the group lacking dynein arms (LODA) (r = 0.91, p < 0.05). Eggs from the groups of patients with LODA and those with antisperm antibodies had thicker zona pellucida than others (p < 0.05). These findings suggest that in addition to nuclear criteria of maturity, the growth of oocytes is an important factor for fertilising ability. Insufficient development of the ooplasm may contribute to fertilisation failure, particularly when sperm with functional defects are used. In contrast, a thick zona pellucida may prevent sperm with specific anomalies such as LODA or antisperm antibodies from penetrating into the perivitelline space.

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

2014 ◽  
Vol 26 (6) ◽  
pp. 847 ◽  
Author(s):  
M. E. Arias ◽  
R. Sánchez ◽  
J. Risopatrón ◽  
L. Pérez ◽  
R. Felmer
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Membrane Integrity ◽  
Control Group ◽  
Developmental Potential ◽  
Bovine Spermatozoa ◽  
Pronuclear Formation ◽  
First Time ◽  
In Vitro Embryonic Development

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

scholarly journals The in vitro effect of kanamycin on the behaviour of bovine spermatozoa

2019 ◽  
Vol 1 (4) ◽  
pp. 36-40
Author(s):  
Michal Ďuračka
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
In Vitro Cultivation ◽  
Sperm Analysis ◽  
Time Dependent Effect ◽  
Bovine Spermatozoa ◽  
And Function ◽  
Vitro Effect

The use of antibiotics is a common part of animal biotechnologies. Especially, the use of antibiotics in semen extenders is necessary. However, the effect of antibiotics on the spermatozoa structure and function is still not completely examined. Therefore, the aim of our study was to investigate the effect of kanamycin on bovine spermatozoa at concentrations of 80 and 160 µg/mL during the 24 h in vitro cultivation. Semen samples were collected from clinically healthy Holstein-Friesian bulls. At times of 0, 2 and 24 h the motility assessment, mitochondrial activity, acrosomal and membrane integrity evaluation were performed. The sperm motility was measured using the Computer-assisted sperm analysis (CASA). Mitochondrial activity was evaluated through the Mitochondrial Toxicity Test (MTT). The acrosomal status was determined using the fast green/rose bengal staining on slides. Similarly, the membrane integrity was analysed using the eosin-nigrosin staining. Our results revealed the dose- and time-dependent effect of kanamycin under the in vitro conditions. In conclusion, the selected concentrations of kanamycin may have adverse effects on the motility, mitochondrial activity, acrosomal and membrane integrity during semen processing. Considering the relatively low concentrations used, we do not recommend to use kanamycin as a supplement in bovine semen extenders.

274 LYSOLECITHIN TREATMENT OF ELAND, BONGO, AND BOVINE SPERMATOZOA AND CLEAVAGE OF BOVINE OOCYTES AFTER INTERSPECIES INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 217
Author(s):  
G. Wirtu ◽  
C. E. Pope ◽  
M. C. Gomez ◽  
R. A. MacLean ◽  
D. L. Paccamonti ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Intracytoplasmic Sperm Injection ◽  
Membrane Integrity ◽  
Oocyte Activation ◽  
Success Rates ◽  
Offspring Production ◽  
Embryonic Cleavage ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

Compared to success rates in human, intracytoplasmic sperm injection (ICSI) is inefficient in ungulate species. Although factors such as injection of membrane-intact sperm and toxic effects of acrosome contents are suspected causes, the reasons for the inefficiency are unclear. A recent report in mice demonstrated that ICSI using spermatozoa treated with a physiological detergent, lysolecithin, improved oocyte activation, cleavage, and offspring production after embryo transfer (Morozumi K et al. 2006 PNAS 109, 17 661–17 666). The objectives of the present study were to evaluate the effects of detergent treatment on motility and membrane integrity of frozen thawed eland, bongo and bovine spermatozoa and to examine sperm decondensation/embryonic cleavage following ICSI of in vitro-matured bovine oocytes. In experiment 1, sperm motility was observed on a warm microscope stage during exposure to 3 lecithin concentrations, 0.04, 0.02, and 0.01%, and the time at which 100% of the spermatozoa lost motility was recorded. In experiment 2, spermatozoa were exposed to 0.02% lecithin for 22 s, and the membrane integrity and acrosome status of spermatozoa were determined using a combined trypan blue-Giemsa staining (Nagy et al. 1999 Theriogenology 52, 1153–1159). In experiment 3, bovine oocytes were injected, using the piezo drill, with lecithin-treated (0.02%, immobilized) or untreated (piezo pulse immobilized) eland, bongo, or bovine spermatozoa and subsequently cultured for 2 days in CR1aa containing 3 mg mL–1 BSA. Each experiment was replicated at least 3 times. Lecithin induced time- and concentration-dependent loss of sperm motility. The average time to loss of motility in 100% of the spermatozoa at 0.04, 0.02, and 0.01% lecithin was 107, 222, and 344 s in bovine; 82, 135, and 179 s in eland; and 65, 115, and 158 in bongo, respectively. Data on membrane integrity (intact or nonintact) and acrosome status (reacted or nonreacted) of detergent-treated or control spermatozoa are shown in Table 1. Sperm head decondensation and embryonic cleavage were observed following homologous and interspecies (antelope into bovine) ICSI of lecithin-treated or control spermatozoa. In conclusion, lecithin treatment induced concentration and time-dependent loss of motility and was effective in damaging the sperm membrane and acrosome in eland, bongo, and domestic bulls. Eland and bongo spermatozoa underwent decondensation and activated bovine oocytes after interspecies ICSI. Table 1.

In Vitro maturation rate of oocytes collected from indigenous cows of Assam after slaughter

2015 ◽  
Vol 49 (6) ◽  
Author(s):  
S. S. Deka ◽  
D. J. Kalita ◽  
S. Sarma ◽  
D. J. Dutta
Keyword(s):  
Zona Pellucida ◽  
Maximum Yield ◽  
Cumulus Cells ◽  
Cumulus Expansion ◽  
Slaughter House ◽  
Oocyte Collection ◽  
Maturation Rate ◽  
The Mean ◽  
Ideal Method

Sixty healthy ovaries were collected from local slaughter house. Oocytes from small and medium sized follicles (2-8 mm in diameter) were selected for oocyte collection using aspiration followed by slicing . A and B category ( cumulus oocyte complexes (COCs) with more than 5 layers and 3-5 layers respectively of compact cumulus cells surrounding the zona pellucida) were selected for <italic>in vitro</italic> maturation. A total of 361 oocytes were found to be matured with overall mean maturation rate of 82.59 ± 0.02%. The mean number of grade A, B and C oocytes recovered per ovary was 4.26 ± 0.53, 3.05 ± 0.31 and 1.25 ± 0.19, respectively. The overall recovery of grade A, B and C oocytes were 256, 183 and 74 . Out of 439 oocytes 58.86 ± 0.05%, 33.23 ±0.04 % and 9.90 ± 0.04 % showed +++, ++ and + degrees of cumulus expansion, respectively. It was concluded that aspiration followed by slicing is an ideal method for maximum yield of oocytes and <italic>in vitro</italic> maturation in indigenous cow of Assam.

scholarly journals The effect of elevated non-esterified fatty acid concentrations on bovine spermatozoa and on oocyte in vitro fertilisation

2018 ◽  
Vol 30 (11) ◽  
pp. 1553
Author(s):  
K. L. J. Desmet ◽  
W. F. A. Marei ◽  
I. Pintelon ◽  
P. E. J. Bols ◽  
J. L. M. R. Leroy
Keyword(s):  
Fatty Acid ◽  
Membrane Integrity ◽  
In Vitro Fertilisation ◽  
Sperm Function ◽  
Treatment Conditions ◽  
Developmental Capacity ◽  
Solvent Control ◽  
Bovine Spermatozoa ◽  
Oviductal Fluid

Elevated non-esterified fatty acid (NEFA) concentrations, present in follicular and oviductal fluid, have been postulated as a causative link between metabolic disorders and subfertility. High NEFA conditions can directly disrupt oocyte maturation and developmental capacity after fertilisation. However, their influence on sperm function and the fertilisation process is not known. This study investigated the fertilisation process under high NEFA conditions. To differentiate between effects on both spermatozoa and oocytes or on spermatozoa only, different experiments were conducted. In the first experiment both gametes were simultaneously incubated during IVF under different conditions: (1) NEFA-free, solvent-free control conditions, (2) solvent control, (3) physiological concentrations of oleic (OA), palmitic (PA) and stearic (SA) acids or (4) pathophysiological concentrations of OA, PA and SA. In the second experiment spermatozoa were incubated (4 h) under the same treatment conditions prior to routine IVF. Gamete co-incubation resulted in reduced fertilisation and cleavage rates and increased prevalence of polyspermy. In the second experiment embryo developmental capacity and quality were not affected, although sperm motility and plasma membrane integrity were decreased. In conclusion, lipolytic conditions affected the fertilisation process mainly through an effect on the oocyte. Spermatozoa were still able to fertilise even though these conditions reduced sperm function.

scholarly journals Effect of Addition of Melatonin on the Liquid Storage (5°C) of Mithun (Bos frontalis) Semen

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
P. Perumal ◽  
Kezhavituo Vupru ◽  
K. Khate
Keyword(s):  
Membrane Integrity ◽  
Control Group ◽  
Protective Effects ◽  
Sperm Abnormality ◽  
Total Antioxidant ◽  
Biochemical Profiles ◽  
Group 5 ◽  
Group 2

The present study was undertaken to assess the effect of melatonin (MT) on sperm motility, viability, total sperm abnormality, acrosomal and plasma membrane integrity, DNA abnormality, antioxidant profiles such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total antioxidant capacity (TAC), enzymatic profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT), and biochemical profiles such as malonaldehyde (MDA) production and cholesterol efflux. Total numbers of 30 ejaculates were collected twice a week from eight mithun bulls and semen was split into five equal aliquots, diluted with the TEYC extender. Group 1 has semen without additives (control) and group 2 to group 5 have semen that was diluted with 1 mM, 2 mM, 3 mM, and 4 mM of melatonin, respectively. These seminal parameters, antioxidant, enzymatic, and biochemical profiles were assessed at 5°C for 0, 6, 12, 24, and 30 h of incubation. Inclusion of melatonin into diluent resulted in significant (P<0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa, and acrosomal abnormalities at different hours of storage periods as compared with control group. Additionally, melatonin at 3 mM has significant improvement in quality of mithun semen than melatonin at 1 mM, 2 mM or 4 mM stored inin vitrofor up to 30 h. It was concluded that the possible protective effects of melatonin on sperm parameters are it prevents MDA production and preserve the antioxidants and intracellular enzymes during preservation.

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