87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

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Sperm penetration of in vitro bovine oocytes with sperm or oocytes preloaded with hoechst 33342 fluorochrome

Theriogenology ◽

10.1016/s0093-691x(98)90648-3 ◽

1998 ◽

Vol 49 (1) ◽

pp. 295

Author(s):

L.M. Olsen ◽

G.L. Woods ◽

C.S. Schneider ◽

R.W. Wright

Keyword(s):

Hoechst 33342 ◽

Sperm Penetration ◽

Bovine Oocytes

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

Zygote ◽

10.1017/s0967199401001095 ◽

2001 ◽

Vol 9 (1) ◽

pp. 89-95 ◽

Cited By ~ 3

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.

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In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Theriogenology ◽

10.1016/0093-691x(91)90380-v ◽

1991 ◽

Vol 36 (2) ◽

pp. 209-219 ◽

Cited By ~ 11

Author(s):

R.C. Chian ◽

K. Niwa ◽

K. Okuda

Keyword(s):

Zona Pellucida ◽

Before And After ◽

Bovine Oocytes

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Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

Zygote ◽

10.1017/s0967199403001096 ◽

2003 ◽

Vol 11 (1) ◽

pp. 69-76 ◽

Cited By ~ 21

Author(s):

S.A. Ock ◽

J.S. Bhak ◽

S. Balasubramanian ◽

H.J. Lee ◽

S.Y. Choe ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Chromosomal Abnormality ◽

Polar Body ◽

Ultrastructural Changes ◽

Time Interval ◽

Oocyte Activation ◽

Group 4 ◽

Group 5 ◽

Bovine Oocytes

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

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Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

Theriogenology ◽

10.1016/s0093-691x(02)00695-7 ◽

2002 ◽

Vol 57 (8) ◽

pp. 2093-2104 ◽

Cited By ~ 4

Author(s):

Byung-Ki Kim ◽

Sang-Chan Lee ◽

Kwang-Sun Lee ◽

Bok-Kyu Lee ◽

Chang-Hee Han ◽

...

Keyword(s):

Sperm Penetration ◽

Pronuclear Formation ◽

Bovine Oocytes

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119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab119 ◽

2012 ◽

Vol 24 (1) ◽

pp. 172 ◽

Cited By ~ 1

Author(s):

M. J. Sanchez-Calabuig ◽

P. Beltran-Brena ◽

E. Martinez-Nevado ◽

D. Rizos ◽

J. F. Perez-Gutierrez ◽

...

Keyword(s):

Bottlenose Dolphin ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Sperm Chromatin ◽

Cleavage Rate ◽

Vitro Fertilization ◽

Sperm Penetration ◽

Pronuclear Formation ◽

Bovine Oocytes

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

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376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab376 ◽

2007 ◽

Vol 19 (1) ◽

pp. 303

Author(s):

M. Nakai ◽

N. Kashiwazaki ◽

N. Maedomari ◽

M. Ozawa ◽

J. Noguchi ◽

...

Keyword(s):

Freeze Drying ◽

Oocyte Activation ◽

Sham Injection ◽

Oocyte Meiosis ◽

Freeze Dried ◽

Sperm Penetration ◽

Sperm Factor ◽

Pre Treatment ◽

Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

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Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes

Microscopy and Microanalysis ◽

10.1017/s1431927619000692 ◽

2019 ◽

Vol 25 (4) ◽

pp. 1032-1036 ◽

Cited By ~ 1

Author(s):

Francisco Báez ◽

Álvaro A. Camargo ◽

Gustavo D.A. Gastal

Keyword(s):

Zona Pellucida ◽

Ultrastructural Change ◽

Early Embryo ◽

Imaging Analysis ◽

Mature Group ◽

Immature Group ◽

Imagej Software ◽

Bovine Oocytes ◽

Objective Methodology

AbstractThe aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

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