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Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes/Pre-incubacao dos espermatozoides suinos diminui polispermia e aumenta a producao embrionaria em oocitos de baixa qualidade.

INTRODUCTION

The embryo IVP technique can be employed on different segments of assisted reproduction in humans and animals (DYCK et al., 2014), allowing a better understanding of maturation, fertilization, sperm capacitation, embryo development and their regulatory mechanisms (COY & ROMAR, 2002). Since the first embryos obtained from in vitro fertilization (IVF) in pigs (MATTIOLI et al., 1989), the process experienced a great evolution. However, the porcine in vitro embryo development is still lower than the in vivo (KWAK et al., 2014). One of the determining factors for the low efficiency of porcine embryo IVP is the elevated rate of polyspermy (TOKESHI et al., 2007; FAUSTINI et al., 2010). Great variations have been observed in polyspermy rates of both fresh (SIRARD et al., 1993) and frozen semen (NAGAI et al., 1988). Polyspermy rates are directly related to the sperm number at the site of fertilization (FAUSTINI et al., 2010), and also with the oocyte quality (SUZUKI et al., 2003). It is believed that the oviduct microenvironment participates in polyspermic blocking during in vivo fertilization (LUNO et al., 2013). The in vivo incidence of polyspermy is less than 10% (GIOIA et al., 2005). However, in vitro matured oocytes are known to show a lower cortical reaction, and an erroneous distribution of the granules around the oocyte. This failure of the IVC systems results in a poor release of cortical granules after sperm penetration, thus resulting in failure of the zona pellucida reaction, and consequently in polyspermy (FUNAHASHI et al., 2000). These ratios are even greater in low quality oocytes (SUZUKI et al., 2003). In porcine IVF procedures performed with slaughterhouse ovaries it is possible to obtain a great number of oocytes. However, after classification, approximately 10% of these oocytes are classified as of excellent quality (data not shown), demanding a great number of ovaries, especially when using adult gilts. Therefore, in assisted reproductive procedures (i.e. embryo IVP) where the goal is to preserve endangered pig breeds like Moura, Caruncho, Piau and others, the use of exclusively high/excellent quality oocytes might not be possible. Moreover, in procedures such as oocyte cryopreservation, the standard protocols per se negatively affect the gamete quality. Ultimately, the search for strategies that improve the number of blastocysts obtained out from a pool of low quality oocytes is very important.

MATERIALS AND METHODS

Experimental design

The first experiment was performed in order to assess embryo production from low versus high quality oocytes produced by a standard porcine IVF protocol. In experiment 1, the oocytes of high quality (n=163) and low quality grade (n=143) were matured, fertilized and cultured under identical conditions in order to assess the rates of embryonic development (5 replications). Aiming to improve the IVF efficiency using low quality oocytes, experiment 2 (n=1454) assessed cleavage and blastocyst rates, as well as cell density. Oocytes were in vitro matured and fertilized with sperm previously incubated during different periods: 0h (control), 0.5h, 1h and 1.5h. These results were compared to a positive control using oocytes of high quality (7 replications). In order to better understand the results obtained in the second experiment, the experiment 3 (n=619) aimed to assess fertilization and monospermy rates after IVF of low quality oocytes. To that, oocytes were in vitro matured and fertilized with sperm pre-incubated for different periods: 0h (control), 0.5h, 1.5h and 1h, being these results compared to a positive control of high quality oocytes (5 replications). The fourth experiment aimed to assess whether semen pre-incubation would improve the blastocyst rates for high quality oocytes. To that, experiment 4 evaluated the use of semen pre incubation for 1.5h (pointed in experiment 2) in oocytes of high quality. The embryo development, cell density, fertilization and monospermy rates (5 replications) were assessed.

Chemicals and reagents

Except where indicated, all chemicals used came from Sigma Chemical Co. (St. Louis, MO, USA).

Oocytes search and selection

Ovaries of peri-pubertal gilts obtained at abattoirs were kept in saline solution at 30 to 35[degrees]C, and transported to the laboratory within 5 hours. The cumulus oocyte complexes (COCs) were aspirated from 3 to 6mm diameter follicles, with the aid of a vacuum line at a rate of 15mL of water flow per minute, and stored in 15mL conic tubes. After 15 minutes, the sediment from each tube was deposited into a 90mm Petri dish containing centrifuged follicular fluid, to search the CCOs, under stereomicroscope. The COCs were classified as high or low quality, as follows: High quality CCOs had at least three layers of compact cumulus cells and homogeneous cytoplasm, whereas the low quality COCs were characterized by gaps in coverage of cumulus cells, as well as an heterogeneous cytoplasm due to the presence of vesicles or granules.

Oocyte maturation

All steps of embryo in vitro production were performed in NUNC 4-well dishes (Nunclon, Roskilde, Denmark). For maturation, 40 to 50 COCs were placed into wells containing 400 [micro]L of TCM199 medium supplemented with 26.19mM Sodium Bicarbonate, 25% gilts Follicular Fluid, 0.1mg [mL.sup.-1] L-cysteine, 10ng [mL.sup.-1] Epidermal Growth Factor, 2.19mM Sodium Pyruvate, 3.05mM D-glucose, 0.5mg [mL.sup.-1] Luteinizing Hormone (LH) (Lutropin, Bioniche), 0.01UI [mL.sup.-1] Follicle Stimulating Hormone (FSH) (Folltropin, Bioniche), 1mM dibutirilAMPc (dbcAMP), 100IU [mL.sup.-1] Penicillin G and 0.1mg [mL.sup.-1] Streptomycin Sulfate. The COCs remained in this medium for 22 hours at 38.8[degrees]C, in an atmosphere of 5% C[O.sub.2] and saturated humidity. Afterwards, oocytes were transferred to a second maturation medium, similar to the above but without LH, FSH, and dbcAMP, where they remained for another 18 to 22 hours under the same culture conditions.

Sperm Collection and Selection

Freshly ejaculated semen was obtained from a boar previously tested for IVF, and proven to produce IVF embryos in a regular rate. The ejaculate was mixed with BTS (Beltsville Thawing Solution) extender containing 41.5g [mL.sup.-1] D-glucose, 5.9g [mL.sup.-1] Sodium Citrate (Merck), 0.5g [mL.sup.-1] Sodium Bicarbonate, 0.8g [mL.sup.-1] EDTA, 0.3g [mL.sup.-1] Potassium Chloride and 1mg [mL.sup.-1] Gentamicin Sulfate. Semen was diluted 1:3 in BTS and maintained in a temperature of 15 to 17[degrees]C, being gently mixed every six hours, until IVF. Each ejaculate was obtained and processed for IVF within maximum 18 hours after collection. Prior to use, semen was incubated at 38.8[degrees]C for 10 minutes and the sperm selection for IVF was then performed by centrifuging (13.100 x g/ two minutes) 150 [micro]L of semen through discontinuous Percoll gradient (45 and 90%, respectively). The selected sperm cells were then mixed in 1000 [micro]L of m-TBM consisting of 113.1mM Sodium Chloride, 3.0mM Potassium Chloride, 7.5mM Calcium Chloride, 20mM Tris (Merck), 11mM D-glucose, 5mM Sodium Pyruvate, supplemented with 2mg [mL.sup.-1] Bovine Serum Albumin (BSA) and centrifuged again for 30 seconds at 11.400 x g. Semen was then diluted in 5mL m-TBM, and sperm concentration was determined with the aid of a Neubauer chamber.

Pre-incubation

Spermatozoa were transferred to 4 well dishes with 400 [micro]L of m-TBM supplemented with 0.4mg [mL.sup.-1] caffeine and 2mg [mL.sup.-1] BSA in a concentration of 62,500 spermatozoa [mL.sup.-1]. According to the experimental groups, sperm samples were preincubated in the fertilization medium for different periods: 0 hour (control), 0.5, 1 or 1.5h at 38.8[degrees]C, under a 5% C[O.sub.2] atmosphere and saturated humidity. The semen of all groups came from the same ejaculate.

In vitro fertilization (IVF)

The matured COCs were mechanically denuded in TCM-199 added with 2.19mM Sodium Pyruvate, 100IU [mL.sup.-1] Penicillin G, 0.1mg [mL.sup.-1] Streptomycin Sulfate, and 20% Fetal Bovine Serum (FBS) (Nutricell--SP). Just after, the oocytes were transferred to a dish containing the pre-incubated spermatozoa, remaining for 3 hours in co-incubation.

IVF assessment

After IVF, the sperm adhered to zona pellucida were removed by successive pipetting. Twelve hours after fertilization, approximately one third part of the structures were fixed in absolute Ethanol (Labsynth, Brazil) supplemented with 0.01mg [mL.sup.-1] Bisbenzimide Trihydrochloride H-33342. The fixed and stained structures were placed between slide and cover slip and exposed to UV light for visualization and counting of male and female pronuclei as well as first and second polar bodies. Structures were classified as non-fertilized (absence of sperm and/or male pronucleus); correctly fertilized (presence of two polar bodies and two (male and female) pronuclei); or improperly fertilized (presence of polyspermy, of more than two pronuclei).

In vitro culture

After IVF excessive sperm were removed and presumptive zygotes were washed and cultured in PZM-3 (Porcine Zygote Medium 3) (YOSHIOKA et al., 2002) during four days (D0 = fertilization) at 38.8[degrees]C under an atmosphere of 5% C[O.sub.2] and saturated humidity. On fourth day of culture 10% FCS was added to the medium. Cleavage and embryo development rates were assessed at day two (cleavage) and day seven (blastocysts) of culture.

Embryo quality assessment

Blastocysts at day seven of culture were fixed in absolute Ethanol (Labsynth, Brazil) supplemented with 0.01mg [mL.sup.-1] Bisbenzimide Trihydrochloride H-33342 for at least 15min. The fixed and stained structures were placed in a 2[micro]L glycerol drop between slide and cover slip and exposed to UV light for visualization. The stained nuclei of the embryo cells were counted as an estimative of embryo cell density.

Statistical analysis

Data were analyzed using the JMP software version 5, 2002 (SAS INSTITUTE INC. CARY, NC, USA). Cleavage and blastocyst development were analyzed by the chi-square test. All remaining data, including fertilization and monospermic penetration rates, IVF efficiency and blastocysts total cell number were analyzed by ANOVA and compared through the Tukey test. For fertilization and monospermic penetration rates as well as for and IVF efficiency, data were normalized by the square root of the value divided by 100. Percentages of development to the blastocyst stage were based on the number of oocytes cultured. The fertilization rates and IVF efficiency were based in the total of matured oocytes. Monospermic penetration rates were calculated with basis on the total of fertilized oocytes. Level of significance for all analyses was 5%.

RESULTS

In the experiment 1, oocytes of high quality presented greater cleavage (74.8%) and blastocyst (33.7%) rates (P<0.05) than their low quality counterparts (51.7 and 9.8%, respectively). However, no differences were observed in the embryo cell density, with 66.7[+ or -] 3.7 for the high quality versus (68.5 [+ or -] 7.1) for the low quality oocytes.

In the second experiment, among the low-quality oocytes, all pre-incubation periods (0.5h, 1h and 1.5h) resulted in an increase in the cleavage rate, in comparison with the absence of pre-incubation. The greatest cleavage rate (92.5%) among all the groups was observed with 1.5h pre-incubation time (Table 1). Regarding the embryo development to the blastocyst stage, it was observed a significant improvement with a longer (1.5h) incubation period, when low quality oocytes were used. However, it did not reach the rates observed with high quality oocytes in the control group (Table 1).

In the Experiment 3, although the fertilization rate was similar among all groups (P>0.05), the rate of monospermy in the group pre incubated for 1.5h was the highest (P<0.05) amongst all groups (Table 2). The highest monospermy rate resulted in an increase of IVF efficiency, which was similar to the oocytes with high quality (control group).

In the experiment 4, the pre-incubation of spermatozoa did not affect (P [greater than or equal to] 0.05) the cleavage (76.1 vs. 75.3%) and blastocyst (30.5 vs. 24.7%) rates for oocytes of high quality, nor influenced the cell density in embryos (66.7[+ or -]3.5 vs 72.0[+ or -]3.7). Furthermore, it has also been shown that semen preincubation does not affect the fertilization rates (64.9 vs. 75.3%) or monospermic penetration rates (44.3% vs 48.4/n=179) in fertilization of high quality porcine oocytes.

DISCUSSION

Although high polyspermic penetration rates are well documented in pigs (FUNAHASHI 2003; SUZUKI et al., 2003), some interesting interactions of oocyte quality and the sire have been reported in cattle (OHLWEILER et al., 2013), where depending on the technique used to artificially fertilize the oocyte, the embryo development rate was affected or not by the oocyte quality. Unfortunately, oocytes of low quality are predominant in gilt ovaries. In this study, among all oocytes classified for the experiments, the ones of excellent quality were as low as around 10% of the total. This low efficiency was confirmed in the experiment 1 of this study, which resulted in a blastocyst yield of 33.7% with high quality oocytes vs. 9.8% for the ones of low quality. It has been already established that low quality oocytes have poor maturation and deficient cortical reaction at the time of fertilization, which leads to a lower embryo production (SUZUKI et al., 2003). NAGAI et al. (1996) suggested that to develop practical IVM-IVF protocols in pigs it is necessary to stablishe reliable methods to synchronize the fertilizing capacity of sperm and fertility of the oocytes. So, as alternative to obtain an efficient IVP of porcine embryo is to search for protocol adaptations so that good rates of embryo development could be achieved, even when using low quality oocytes. A key factor for the low efficiency of IVP porcine embryos is the high rate of polyspermy (TOKESHI et al., 2007), which is directly associated to the amount of sperm at the site of fertilization (FAUSTINI et al., 2010), and also the oocytes quality (SUZUKI et al., 2003). When several spermatozoa penetrate the oocyte, the DNA de-condensation is difficult, probably due to intense competition for factors involved in the process of pronucleus formation (COY et al., 1993b). Thus, reduction in the polyspermy rates is highly desirable in the porcine IVP. To overcome the obstacle posed by pig oocytes of low quality, we proposed to adjust the sperm preparation according to the oocyte quality grade. Therefore, by using different semen pre-incubation periods prior to IVF, a positive effect was observed. The semen preincubated during 1.5h resulted in an increase for cleavage and blastocyst rates. Previous reports had already shown that a reduced number of available adequately selected/pre-capacitated sperm during in vitro fertilization results in lower polyspermy rate in pigs (FUNAHASHI & NAGAI 2000). This is probably due to the better recognition of sperm penetration by oocytes, when sperm are properly prepared. We showed an increase in monospermic penetration rates with semen pre incubation during 1.5 hours (experiment 3), without changing in the fertilization rates (Table 2). This shows that the sperm pre incubation does not affect the oocyte penetration rates, but probably does improve the sperm cell recognition by the oocyte, thus triggering the blockage to polyspermy. Even showing better monospermic penetration rates than the high quality oocytes, the IVF of low quality oocytes using pre-incubation of semen for 1.5h did not reach similar embryo development. Conversely, it was demonstrated that high quality oocytes used in IVF were not influenced by semen pre-incubation. This occurs because the oocytes of better quality are more efficient in blocking polyspermy, in comparison to their low quality counterparts (JACKOWSKA et al., 2009). This reinforces that pre incubation does not affect the viability of sperm cells, but only facilitates low quality oocytes recognition for sperm penetration. It should be noted that within the different experiments, no effect has been observed in embryo cell density, evidencing that the blastocyst quality remains the same, despite semen being preincubated or not. This shows that using sperm pre incubation, low quality oocytes were allowed to produce a larger amount of embryos, but without affecting their quality. However, there is clear evidence that pre-incubation of sperm cells during 1.5h before IVF procedures is a good strategy to improve embryo developing rates out of a pool of low quality porcine oocytes.

CONCLUSIONS

For low quality oocytes, the blastocyst rates are improved after semen pre-incubation for 1.5h. Overall, semen pre-incubation for 1.5h improved monospermy in low quality oocytes, even in comparison to high quality oocytes fertilized with not pre-incubated semen. Nonetheless, blastocyst rates for oocytes of high quality are not influenced by semen pre incubation during porcine IVF procedures.

http://dx.doi.org/10.1590/0103-8478cr20150700

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Claudio Francisco Brogni (I) Lain Uriel Ohlweiler (I) Norton Klein (I) Joana Claudia Mezzalira (I) Jose Cristani (I) Alceu Mezzalira (I) *

(I) Laboratorio de Reproducao Animal prof. Assis Roberto de Bem, Universidade do Estado de Santa Catarina (UDESC), 88520-000, Lages, SC, Brasil. E-mail: alceu.mezzalira@udesc.br. * Corresponding author.

Received 05.15.15 Approved 11.07.15 Returned by the author 03.14.16 CR-2015-0700.R2
Table 1--Cleavage and blastocyst rates and cellular
density obtained after embryonic in vitro production
from porcine oocytes of high and low quality grade,
without pre-incubation (0h) or with different periods
of sperm pre-incubation (Experiment 2).

Group       Total of        Cleavage
           structures
                         n        %

High 0h       265       210   79.2 (b)
Low 0h        361       157   43.5 (d)
Low 0.5h      346       243   70.2 (d)
Low 1h        386       283   73.3 (bc)
Low 1.5h      361       334   92.5 (a)

Group        Blastocyst         Cell number

           n       %

High 0h    67   25.3 (a)   66.3 [+ or -] 3.5 (a)
Low 0h     27   7.5 (c)    68.5 [+ or -] 71
Low 0.5h   27   7.8 (c)    68.8 [+ or -] 7.7 (a)
Low 1h     30   7.8 (c)    58.7 [+ or -] 6.9 (a)
Low 1.5h   54   15.0 (b)   71.3 [+ or -] 4.9 (a)

Different letters in the same column differ
statistically (P < 0.05).

Table 2--Fertilization, monospermic index and efficiency
of IVF in oocytes of low and high quality, fertilized with
semen without pre-incubation (0h) or with pre-incubation
(Experiment 3).

Group       Total of    Fertilized       Monospermic        IVF
           structures                                    efficiency

                         n       %       n       %           %

High 0h        70       47    67.1 (a)   18   38.3 (b)   25.7 (ab)
Low 0h        131       86    65.6 (a)   27   31.4 ()    20.6 (b)
Low 0.5h      150       118   78.7 (a)   46   39.0 (b)   30.7 (ab)
Low 1h        136       98    72.1 (a)   27   27.6 (b)   19.9 (b)
Low 1.5h      132       105   79.5 (a)   56   53.3 (a)   42.4 (a)

Different letters in the same column differ
statistically (P [less than or equal to] 0.05).
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Title Annotation:produccion animal; texto en ingles
Author:Brogni, Claudio Francisco; Ohlweiler, Lain Uriel; Klein, Norton; Mezzalira, Joana Claudia; Cristani,
Publication:Ciencia Rural
Date:Jun 1, 2016
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