378 INVOLVEMENT OF THE SPERM TAIL IN TRIGGERING OOCYTE ACTIVATION IN PIGS

2010 ◽  
Vol 22 (1) ◽  
pp. 346
Author(s):  
M. Nakai ◽  
J. Ito ◽  
K. Sato ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Sperm Head ◽  
Antigenic Peptide ◽  
Oocyte Activation ◽  
Tail Region ◽  
Sperm Tail ◽  
Sperm Factor ◽  
Percoll Density Gradient ◽  
Strong Candidate ◽  
University Of Massachusetts

In mammals, repetitive increases of the intracellular Ca2+ level, known as Ca2+ oscillations, are observed in oocytes immediately after sperm-oocyte fusion, which is a prerequisite event for oocyte activation. Previous studies indicate that phospholipase C zeta (PLCζ), a strong candidate sperm factor for triggering Ca2+ oscillations, is localized in the sperm head of several mammalian species. We have reported that the rate of pronucleus formation in oocytes injected with a sperm head is lower than that for oocytes injected with a whole spermatozoon (Nakai et al. 2009 IETS). This has given rise to a hypothesis that not only the sperm head but also the tail play a role in inducing oocyte activation in pigs. In this study, we attempted to detect the localization of PLCζ in the pig sperm tail and also its ability to activate porcine oocytes after injection. To clarify the localization of PLCζ in pig sperm, frozen-thawed ejaculated pig sperm were immunostained using an anti-PLCζ antibody that has been reported previously (Kurokawa et al. 2005). Western blotting was also carried out to examine whether PLCζ (72 kDa) was present in the sperm tail. Sperm tails were detached from the head by sonication and then collected after centrifugation in a Percoll density gradient. We also confirmed whether the sperm tail itself had the ability to trigger oocyte activation using the following 4 injection groups: (1)1 sperm head (Head), (2) 1 sperm tail (Tail), (3) 1 sperm head and 1 tail (Head + Tail), and (4) Sham. The nuclear status of the injected oocyte was evaluated at 10 h after injection. In the present study, we used 3 sperm samples that were prepared from different boars. In pig sperm, the acrosome, tail, and post-acrosomal regions were stained by the PLCζ antibody. The signals in both the post-acrosomal and tail regions disappeared after pretreatment with antigenic peptide, but that in the acrosome region was retained. Furthermore, we confirmed the presence of a band of approximately 72 kDa from the sperm tail and also confirmed its disappearance upon pretreatment with antigenic peptide. The rates of oocytes released from metaphase-II arrest in the Head, Tail, and Head+Tail groups were significantly higher than that in the Sham group (P < 0.05 by ANOVA andTukey test). However, most of the oocytes in the Tail group failed to form pronuclei and showed other meiotic stages (anaphase-II, telophase-II, or metaphase-III). In conclusion, we have shown that PLCζ is expressed in the post-acrosomal and tail region of pig sperm. It is suggested that, in the pig, the sperm tail participates in the triggering of oocyte activation. The authors thankRafaelA. Fissore (Department ofVeterinary and Animal Sciences, University of Massachusetts Amherst) for providing the antigenic peptide for PLCζ. This study was supported in part by JSPS Fellowship (71310042 to M.N.) from the Japanese Society for Promotion of Science (JSPS).

scholarly journals Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes

Reproduction ◽  
2002 ◽  
pp. 611-623 ◽  
Author(s):  
LJ Cox ◽  
MG Larman ◽  
CM Saunders ◽  
K Hashimoto ◽  
K Swann ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Mammalian Species ◽  
Blastocyst Stage ◽  
Egg Activation ◽  
Oocyte Activation ◽  
Cynomolgus Monkeys ◽  
Mammalian Oocyte ◽  
Mouse Oocytes ◽  
Sperm Factor ◽  
Mouse Egg

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 157-163 ◽  
Author(s):  
Liliana T. Colombero ◽  
Maureen Moomjy ◽  
E. Scott Sills ◽  
Zev Rosenwaks ◽  
Gianpiero D. Palermo
Keyword(s):  
Structural Integrity ◽  
Polar Body ◽  
Sperm Head ◽  
High Rate ◽  
Human Embryos ◽  
Fish Analysis ◽  
Oocyte Activation ◽  
Sperm Tail ◽  
Early Human

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a ‘sum of parts’ is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.

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.

scholarly journals The effects of inbreeding on sperm morphometry of captive-bred endangered mammals

2017 ◽  
Vol 95 (8) ◽  
pp. 599-606 ◽  
Author(s):  
M. Lawrence ◽  
G. Mastromonaco ◽  
K. Goodrowe ◽  
R.M. Santymire ◽  
W. Waddell ◽  
...  
Keyword(s):  
Captive Breeding ◽  
Negative Relationship ◽  
Tail Length ◽  
Poor Quality ◽  
Sperm Head ◽  
Head Width ◽  
Canis Rufus ◽  
Sperm Tail ◽  
Sperm Morphometry ◽  
Red Wolves

Captive breeding is used for the conservation of endangered species, but inbreeding can result when a small number of founders are used to establish populations. Inbreeding can reduce the proportion of normal sperm in an ejaculate, but may also have effects on sperm size and shape (morphometry). We investigated the effects of inbreeding on sperm morphometry of black-footed ferrets (Mustela nigripes (Audubon and Bachman, 1851)) and red wolves (Canis rufus Audubon and Bachman, 1851) from captive breeding programs to determine if more inbred males produced sperm of poor quality (bulky head, small midpiece, short tail). We measured sperm head length, head width, midpiece length, midpiece width, and tail length on 10 sperm from each male of both species. A negative relationship between variation in sperm tail length and inbreeding coefficient (f) was found in black-footed ferret, suggesting that more inbred individuals will have reduced genetic and phenotypic variation. Analyses indicated a negative relationship between sperm head width and f and a positive relationship between sperm tail length and f in red wolf, suggesting that more inbred male red wolves could have faster sperm. These results indicate that inbreeding affects functionally important aspects of sperm morphometry, but that these effects may not be entirely negative.

P–214 Effect of artificial activation of oocytes (AOA-ICSI) on the ploidy status of the resultant blastocysts. A sibling-oocytes pilot study

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Seo. Pe. Yin
Keyword(s):  
Pilot Study ◽  
Fertilization Rate ◽  
Control Group ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Factor Deficiency ◽  
Sperm Factor ◽  
Blastocyst Rate ◽  
Artificial Activation ◽  
Ploidy Status

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (&lt;30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA

scholarly journals Lack of dynein arms in immotile human spermatozoa.

1975 ◽  
Vol 66 (2) ◽  
pp. 225-232 ◽  
Author(s):  
B A Afzelius ◽  
R Eliasson ◽  
O Johnsen ◽  
C Lindholmer
Keyword(s):  
Oxygen Consumption ◽  
Lactic Acid ◽  
Genetic Disorder ◽  
Lactic Acid Production ◽  
Sperm Head ◽  
Conventional Model ◽  
Fibrous Sheath ◽  
Sperm Tail ◽  
Ultrastructural Appearance ◽  
Dynein Arms

Sermatozoa from two brothers who are not twins were found to be straight and immotile. Examinations of the sperm showed that oxygen consumption and lactic acid production were normal; viability tests showed that the percentage of dead sperm was not increased. The ultrastructural appearance of the sperm tail was normal except for a complete lack of dynein arms and some irregularities in the arrangement of the accessory fibers and the longitudinal columns of the fibrous sheath. The mitochondrial apparatus and the sperm head conform to the conventional model. According to the sliding-filament hypothesis first proposed by Afzelius (1959. J. Biophys. Biochem. Cytol. 5:269.), the arms are responsible for the bending movements of the tail. The simplest explanation for the simultaneous lack of arms and sperm motility appears to be that the two brothers have a genetic disorder involving production, assembly, or attachment of the dynein arms.

Interactions between gametes leading to fertilization: the sperm's eye view

1995 ◽  
Vol 7 (4) ◽  
pp. 927 ◽  
Author(s):  
BT Storey
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Mammalian Species ◽  
Sperm Chromatin ◽  
Peptide Backbone ◽  
Mouse Sperm ◽  
Gamete Interactions ◽  
Sperm Plasma Membrane ◽  
Sperm Penetration ◽  
Strong Candidate

Sexual reproduction requires that the gamete carrying the male-derived haploid chromatin join with the gamete carrying the female-derived haploid chromatin during fertilization to produce the diploid zygote. To accomplish this feat, the sperm must not only meet the egg, it must recognize the egg and be recognized in turn by the egg, and in the end must enter and be engulfed by the egg. In this selective overview of gamete interactions that lead to fertilization, encounters of three kinds, followed by the finale of gamete fusion, are considered from the sperm's viewpoint, with particular emphasis on the mammalian species with the mouse as the principal model. The first encounter is with the zona pellucida of the egg, to whose surface the sperm must bind. Mouse sperm appear to have four binding sites for zona ligands. Three interact with sugar moieties of the oligosaccharide chains of the mouse zona glycoprotein ZP3; the fourth binds a peptide backbone arginine. Capacitation is not required for this encounter, but is obligate for the second encounter--induction of the acrosome reaction in the bound sperm. The acrosome reaction is an exocytotic process that makes available the enzymatic machinery needed for sperm penetration the zona which is the end point of a sequence of reactions directed by intracellular signalling systems. In mouse sperm, these systems are presumed to be activated by ligands on ZP3 binding to ligand-specific sperm receptors with consequent aggregation of receptors. No receptor has been identified with certainty, nor have candidates for putative ZP3 ligands been identified. Completion of the acrosome reaction allows the sperm to penetrate the zona and, bind to the egg plasma membrane, thereby completing the third encounter. In the mouse, a 94-kDa protein appears essential for this binding. In the guinea-pig, a sperm plasma membrane protein (formerly PH-30, now fertilin), is a strong candidate for the mediator of the fusion process by which the egg engulfs the sperm. Decondensation of the sperm chromatin reverses the remarkable packing of DNA organized by sperm protamines. Mitochondrial DNA is also engulfed by the egg; the question of whether this DNA makes a small finite, or null, contribution to cytosolic inheritance is still in debate. The puzzles attending these encounters are presented as reminders of the intricacy and fascination, as well as of the vital necessity, of gamete interaction.

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

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 &lt; 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

276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS

2013 ◽  
Vol 25 (1) ◽  
pp. 286
Author(s):  
J. Ito ◽  
E. Yuhara ◽  
A. Nakamura ◽  
N. Kashiwazaki
Keyword(s):  
Phospholipase C ◽  
Mammalian Species ◽  
Treated Group ◽  
Round Spermatid ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Domestic Species ◽  
Electrical Pulses ◽  
Group 2 ◽  
Pronuclear Formation

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P < 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.

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