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The effect of sperm preparation and co-incubation time on in vitro fertilization of bos indicus oocytes
Animal Reproduction Science 69 (2002) 15–23
The effect of sperm preparation and co-incubation time on in vitro fertilization of bos indicus oocytes M.A.N. Dode a,∗ , N.C. Rodovalho a , V.G. Ueno a , C.E. Fernandes b a b
Embrapa Gado de Corte, Rodovia Br 262 km 4, Caixa Postal 154, CEP, Campo Grande 79002-970, MS, Brazil Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brazil
Received 14 May 2001; received in revised form 29 August 2001; accepted 17 September 2001
Abstract The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm–oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18 h. After co-incubation oocytes were transferred to the culture medium and culture for 44 h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P < 0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm–oocyte co-incubation time was observed (P > 0.05). However, sperm–oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P < 0.05) progressively at 6 h (63.3 and 54.4%) and 12 h (77.6 and 67.6%). No differences (P > 0.05) were observed between 12 and 18 h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P > 0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12 h, and the prolongation of that time for up to 18 h had no detrimental effect on fertilization. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Spermatozoa; Cleavage; Sperm selection; Sperm–oocyte co-incubation time
∗ Corresponding author. Tel.: +55-67-768-2150; fax: +55-67-768-2150. E-mail address: [email protected] (M.A.N. Dode).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 4 8 - 8
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1. Introduction Sperm selection methods are routinely applied on the in vitro fertilization (IVF) system for various species. These methods are used in order to remove seminal plasma/cryoprotectant and to increase sperm quality characteristics (Parrish et al., 1995; Rodriguez-Martinez et al., 1997). Several methods for sperm selection have been described, such as washing/centrifugation (Fukuda et al., 1990), density gradient centrifugation (Parrish et al., 1995), deferential filtration through a glass wool column (Stabbings and Wasik, 1991) and self-migration procedure (Lonergan et al., 1994). The most widely used of these methods are the swim-up and the Percoll centrifugation (Rodriguez-Martinez et al., 1997; Palomo et al., 1999), which are a self-migration and a density gradient procedure, respectively. The Percoll gradient provides a much higher recovery of motile spermatozoa compared to the swim-up (Parrish et al., 1995) and more successful IVF results. However, there is a variation among Percoll batches and some batches can be deleterious to spermatozoa affecting cleavage and blastocyst rates (Avery and Greve, 1995). On the other hand, the swim-up shows a lower recovery rate, but the sperm quality is variable compared to the Percoll method (Parrish et al., 1995; Correa and Zavos, 1996; Rheingantz et al., 2000). Sperm preparation for IVF can also be done by washing/centrifugation with no deleterious effect on cleavage or blastocyst rates (Avery and Greve, 1995). There are differences in the procedures of how each of those methods select motile from immotile spermatozoa. Centrifugation, used in both methods, can damage sperm with generation of reactive oxygen species (ROS) and peroxidation of sperm membranes, which can bring negative effects on sperm–oocyte fusion (Kim and Parthasarathy, 1998; Watson, 2000). In addition, it has been suggested that the passage of the spermatozoa through the Percoll gradient could induce capacitation (Schweitzer et al., 1996; Rosenkranz and Holzmann, 1997). Therefore, if the capacitation time is reduced in the Percoll gradient compared to the other methods, the co-incubation time could also be reduced avoiding the cytotoxic damage caused by the increased amount of reactive oxygen species (ROS) (Kim et al., 1999). In fact, a reduced exposure of oocyte to spermatozoa favors embryo viability in humans, possibly due to a decrease in potential damage from sperm metabolic waste products (Gianaroli et al., 1996; Dirnfeld et al., 1999). Furthermore, the earlier the spermatozoa are capacitated the earlier the penetration will occur avoiding the aging process of the oocyte, which can also cause abnormal fertilization. The present study was carried out to evaluate the effect of the sperm selection method on penetration and cleavage of Bos indicus in vitro matured oocytes. In addition, it was verified whether the sperm–oocyte co-incubation time affects fertilization. 2. Material and methods Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. Semen samples thawed in a water bath at 37 ◦ C were evaluated for motility and submitted to each one of the different treatments: Percoll, swim-up and washing. At the end of each treatment, the semen samples were used for IVF.
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2.1. Sperm selection procedures In the Percoll technique, an aliquot of 500 l of semen was layered on Percoll gradient consisting of 2 ml of 45 and 90% of Percoll and centrifuged at 700 g for 30 min. The Percoll 90% was prepared using 500 l of sp-Talp 10× in 4.5 ml of Percoll (Parrish and Eid, 1994). The 45% Percoll solution was prepared with 1 ml of 90% Percoll and 1 ml of sp-TALP. After centrifugation the supernant was discarded and the pellet was resuspended with 200 l of sp-TALP. In the swim-up procedure, semen sample was placed in a 15-ml centrifuge tube, in which 1 ml of sp-TALP (Parrish et al., 1998) was added, and incubated at 38.5 ◦ C for 1 h. After incubation, the upper 0.85 ml of the supernatant was collected and transferred to other tube and diluted with sp-TALP to give a final volume of 5 ml. Then, it was centrifuged at 120 g for 10 min. After centrifugation, the supernant was discarded and the pellet diluted with 200 l of sp-TALP. For the washing method, after thawing the semen sample was washed twice at 300 g for 5 min in sp-TALP. After the last centrifugation, the pellet was resuspended with 200 l of sp-TALP. 2.2. In vitro maturation (IVM) Ovaries from Nellore cows (Bos indicus) were collected just after slaughter and transported to the laboratory in saline solution (NaCl 0.9%) supplemented with penicillin G (100 IU/ml) and streptomycin sulfate (100 g/ml) at 30 ◦ C. Oocytes from 2 to 5 mm diameter follicles were aspirated with an 18-gauge needle and pooled in a 15-ml conical tube. After sedimentation, the cumulus oocyte complexes (COC) were recovered and selected by using a stereomicroscope. The good quality COCs were washed three times in maturation medium and transferred to a 200-l drop of maturation medium under paraffin oil and incubated for 24 h at 38.5 ◦ C in 5% of CO2 in air. The maturation medium consisted of TCM-199 supplemented with 10% FCS (v/v), 5.0 g/ml of LH (Sigma L-9773), 0.5 g/ml of FSH (Sigma F-8001) and antibiotics (100 IU/ml of penicillin and 100 g/ml of streptomycin), with 5% CO2 in air at 38.5 ◦ C. 2.3. In vitro fertilization (IVF) and embryo culture For IVF, COCs were washed and transferred to the 150-l drop of fertilization medium. Fertilization medium used was TALP (Parrish et al., 1998) supplemented with penicillamine (2 mM), hypotaurine (1 mM), epinephrine (250 mM) and heparin (10 g/ml). Motile spermatozoa obtained by the various sperm selection methods (Percoll gradient, swim-up and washing) were added into the fertilization drop in a final concentration of 1 × 106 spermatozoa/ml. Spermatozoa and oocytes were co-incubated for 3, 6, 12 and 18 h at 38.5 ◦ C with 5% of CO2 in air. After co-incubation, the zygotes were washed and transferred to the embryo culture medium. The medium was TCM-199, supplemented with 10% FCS (v/v), antibiotics (100 IU/ml of penicillin and100 g/ml of streptomycin) and oviductal cells. Oviduct epithelia cells were prepared on the day of ovary collection. The oviducts, transported
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to the laboratory in sterile saline solution at 4 ◦ C, were washed with PBS and dissected. To recover the cells the lumen contents were washed out of the oviduct down into a centrifuge tube. The cells recovered were washed 3 times by centrifugation with PBS (200 g/5 min) and once with culture medium. The final pellet was resuspended with culture medium and cultured for 1–2 days. At the preparation of the embryo culture, drop only cells with visible cilia movement were added. After different sperm–oocyte co-incubation time, the presumptive zygotes were cultured up to 44 h post insemination. The uncleaved oocytes were fixed and stained with lacmoid (1%) and examined under a phase contrast microscope to determine penetration, pronucleus formation and polyspermy. 2.4. Experimental design A total of 1002 in vitro, matured oocytes were used in a four-replicate experiment. After selection by each one of the methods used, sperm samples were evaluated and the concentration was determined. Semen was then added to the fertilization drop and was co-incubated with the oocytes for various periods of time. At 3, 6, 12 and 18 h post insemination, the oocytes were removed from the fertilization drop, washed and transfer into the embryo culture medium. Cleavage, penetration, pronucleus formation and polyspermy rates were determined 44 h post insemination. 2.5. Statistical analysis All data are expressed in percentage and were examined by analysis of variance, in which the effect of treatment, time of incubation, and interaction between treatments and time of incubation were evaluated. When a statistical significant effect was found the differences among treatments means were determined by least significant differences method at a probability value of 0.05% for all parameters. 3. Results The results of the ANOVA showed that there was no effect of sperm selection method in any of the variables studied. On the other hand, time of co-incubation had a significant effect (P < 0.05) in the percentage of penetration and cleavage. There was no interaction between method of selection and sperm–oocyte co-incubation time. Hence, the data for those variables were presented separately as independent variables. Data for penetration, pronucleus formation, polysperrmy and cleavage rates for each treatment at each time point are presented in Table 1. The comparison among the sperm selection methods showed no differences in the penetration or cleavage rate. Nor did any treatment have any significant effect on the formation of pronuclei, nor on the incidence of polyspermy (Table 2). The penetration rate at the time of the first testing (3 h) was low for all treatment (Table 3). The penetration rate increased from 6 to 12 h, but it remained the same for the following incubation time (12 and 18 h). Co-incubation time did not influence the pronucleus formation
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Table 1 Effect of sperm preparation treatment and time of co-incubation on IVF (means ± S.D.) Treatment
Number of oocytes
Time (h)
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
Swim-up
97 83 95 115 71 75 90 88 75 71 82 60
3 6 12 18 3 6 12 18 3 6 12 18
27.3 ± 4.5 68.4 ± 4.2 91.7 ± 1.0 93.4 ± 0.9 28.2 ± 2.3 68.2 ± 8.7 72.6 ± 5.7 74.3 ± 3.2 23.7 ± 2.7 53.3 ± 8.9 68.6 ± 5.4 69.6 ± 6.0
4.9 ± 1.7 3.1 ± 1.1 2.8 ± 1.3 3.9 ± 0.8 6.4 ± 1.8 2.3 ± 0.8 4.4 ± 0.9 0.9 ± 0.4 2.5 ± 1.3 5.4 ± 2.2 0.0 ± 0.0 2.0 ± 0.7
10.7 ± 1.7 10.6 ± 2.9 7.0 ± 1.4 2.0 ± 0.5 7.7 ± 1.6 4.2 ± 1.7 2.1 ± 0.6 4.9 ± 1.0 2.5 ± 0.8 0.0 ± 0.0 6.9 ± 2.1 3.0 ± 1.1
11.9 ± 1.9 53.8 ± 5.4 76.2 ± 2.8 84.8 ± 1.5 9.9 ± 1.9 61.6 ± 8.1 65.1 ± 4.9 69.2 ± 3.6 17.4 ± 0.9 47.9 ± 8.1 61.6 ± 6.0 61.1 ± 5.6
Percoll
Wash
Table 2 Effect of sperm selection method on fertilization characteristics and cleavage rates (means ± S.D. deviation)a Treatment
Number of oocytes
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
Swim-up Percoll Wash
390 324 288
70.3a ± 6.8 60.8a ± 6.7 53.8a ± 6.8
3.7a ± 1.3 3.5a ± 1.1 2.5a ± 1.2
7.6a ± 1.9 4.7a ± 1.1 3.1a ± 1.2
56.7a ± 7.2 51.5a ± 7.3 47.0a ± 6.5
a
Values with same letters are statistically similar (P > 0.05).
Table 3 Effect of sperm–oocyte co-incubation time on fertilization characteristics and cleavage rates (means ± S.D. deviation)a Time (h)
Number of oocytes
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
3 6 12 18
243 229 267 263
26.5a ± 3.8 63.3b ± 8.0 77.6b ± 5.7 79.1b ± 4.7
4.6a ± 1.8 3.6a ± 1.6 2.4a ± 1.2 2.3a ± 0.8
7.0a ± 1.8 4.9a ± 2.4 5.3a ± 1.7 3.3a ± 1.0
13.1a ± 1.9 54.4b ± 7.6 67.6c ± 5.5 71.8b ± 4.7
a
Values with different letters are statistically different (P > 0.05).
or the polyspermy rate. However, the percentage cleaved embryos gradually increase from 3 to 18 h of sperm–oocyte co-incubation time, although no statistical differences were detected between 12 and 18 h (Table 3). 4. Discussion With the advent of IVF and other assisted reproductive technologies, sperm selection became an important procedure to obtain optimal results. It is needed not only to
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remove seminal plasma or cryoprotector, but also to use the best spermatozoa available from a frozen/thawed semen samples. There are several sperm selection methods and the choice of method depends not only of the yield of motile spermatozoa, but also on its technical complexities, the material and equipment needed and the time costs (Mortimer, 1994). In the present study, we compared a very easy, fast and economic method, the washing with other more expensive and time consuming, the Percoll and the swim-up methods. Several reports indicated that the Percoll (Parrish et al., 1995; Correa and Zavos, 1996; Prakash et al., 1998) and swim-up (Risopatron et al., 1996; Palomo et al., 1999) method yield a qualitative superior sperm sample with significantly more motile, viable and morphologically normal spermatozoa than the washing procedure. In spite of the number of studies in this topic, none of these have verified the effect of the three methods simultaneously, on fertilization of bovine oocytes. In the present study, the different methods to select spermatozoa had similar effect on the in vitro penetration, pronucleus formation, polyspermy and cleavage rates. Similar results were obtained by Seidel et al. (1995) and Palomo et al. (1999), who compared the swim-up and Percoll methods. Risopatron et al. (1996), comparing the migration/sedimentation method and the washing procedure showed that the percentage of oocytes with male pronucleus formation was lower in the washing. However, Avery and Greve (1995) reported that the washing method can be used for preparation of sperm to IVF of bovine oocytes with no detrimental effect on cleavage or blastocyst rates. According to Larsson and Rodriguez-Matinez (2000), the cleavage rate is most suitable to evaluate semen samples than the blastocyst rates, most likely because blastocyst is much more dependent on culture conditions during embryo development than cleavage. Therefore, in the present study, we used cleavage as the end point and no differences were observed among treatments. These results indicate that, under the conditions used, a sufficient number of sperm capable of fertilizing were available, even if they had not been selected as occur in the washing method. Therefore, it is possible that the sperm concentration usually used for IVF can overcome the differences in the sperm quality obtained by different methods to select spermatozoa. It seems that in this case the three methods, studied can be used with no detrimental effect on fertilization. In humans, it has been suggested that short-time exposure of oocytes to spermatozoa improves IVF results (Gianaroli et al., 1996; Quinn et al., 1998; Dirnfeld et al., 1999), mainly because a long-time exposure of oocytes to a large number of spermatozoa would create suboptimal conditions due to excessive generation of ROS (Quinn et al., 1998). These products would induce peroxidation of the membrane lipids reducing membrane fluidity and impairing sperm function (Mortimer, 1994). Therefore, time of co-incubation is also an important aspect to be considered when one wants to increase IVF results. It has been reported that the movement of the spermatozoa in capacitation medium or its passage through the Percoll gradient could rinse out some surface glycoproteins and reduce capacitation time (Rosenkranz and Holzmann, 1997). Based in this assumption, it could be expected that the method used to prepare semen for IVF could alter the capacitation time and the life span of the spermatozoa differently, which could alter time required for fertilization. The results of the present study demonstrated that there was an effect of
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the co-incubation time on fertilization, but no interaction between time and sperm selection method occurred. Thus, the variation, which occurs among the various time periods, was similar for all methods. These results show that if the method influences capacitation, the change is not big enough to affect the co-incubation time required for fertilization. Penetration and cleavage rates were lower when the co-incubation time was 3 h which could be expected since spermatozoa needs at least 4 h to penetrate in vitro matured oocytes (Saeki et al., 1991; Pavlok, 2000). This was confirmed by the significant increase in both penetration and cleavage rates observed at 6 h of co-incubation. Fertilization results continued to increase up to 12 h of incubation demonstrating that regardless the method used there is heterogeneity of the sperm population and that spermatozoa do not complete capacitation simultaneously. Actually, even under the same conditions some spermatozoa from the same ejaculate capacitated faster than others (Yanagimachi, 1994). The incidence of polyspermy has been shown to increase as the time of co-incubation increases (Chian et al., 1992; Sumantri et al., 1997). However, a difference among bulls in terms of sperm capacitation times and acrosome reaction has also been reported (Parrish et al., 1986; Yanagimachi, 1994). In the present study, samples from the same bull were used, and no differences in polyspermy were observed for the various sperm–oocyte co-incubation times. The generation of ROS increases under in vitro conditions using 5% of CO2 in air (Kim et al., 1999), therefore as the time increases more of these products will be produced. In fact, in human, 16 h of incubation can be detrimental to IVF results (Quinn et al., 1998; Dirnfeld et al., 1999) due to excessive generation of ROS and other deleterious products from sperm metabolism into the IVF medium. Unlike in the humans, the incubation for 18 h had no detrimental effect on bovine IVF. It is possible that for the bovine those deleterious products are not high enough during that period to cause a significant effect on fertilization. These can be confirmed by the fact that addition of antioxidant during IVF in bovine (Kim et al., 1999) and porcine (Boquest et al., 1999) did not affected cleavage rates. In fact, although the ROS have many detrimental effects on cells they also have some beneficial effects, such as they are needed for acquisition of fertilizing ability by spermatozoa and hydrogen peroxide is required for induction of capacitation in some species (Blodin et al., 1997; Watson, 2000). Therefore, a delicate balance between the amount of ROS generated and scavenged determines whether the final outcome is detrimental or beneficial (Kim et al., 1999). In the present study, it seems that no alteration in these balance has occurred in 18 h of incubation, since cleavage rates were similar to the 12-h period. However, it is important to point out that there is a variation among bulls in regard to the production of ROS, with some bulls generating excessive amounts of ROS during IVF (Kim et al., 1999). It is possible that the bull used in this study does not produce high amounts of ROS avoiding all the problems caused by these products. In conclusion, it can be stated that under the conditions of these study Percoll, washing and swim-up can be used to prepare sperm for IVF with no detrimental effect on fertilization. In addition, regardless the method used better, fertilization results were obtained when sperm–oocyte were co-incubated for 12 h. However, the prolongation of that time for up to 18 h has no detrimental effect on fertilization.
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The effect of sperm preparation and co-incubation time on in vitro fertilization of bos indicus oocytes M.A.N. Dode a,∗ , N.C. Rodovalho a , V.G. Ueno a , C.E. Fernandes b a b
Embrapa Gado de Corte, Rodovia Br 262 km 4, Caixa Postal 154, CEP, Campo Grande 79002-970, MS, Brazil Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brazil
Received 14 May 2001; received in revised form 29 August 2001; accepted 17 September 2001
Abstract The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm–oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18 h. After co-incubation oocytes were transferred to the culture medium and culture for 44 h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P < 0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm–oocyte co-incubation time was observed (P > 0.05). However, sperm–oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P < 0.05) progressively at 6 h (63.3 and 54.4%) and 12 h (77.6 and 67.6%). No differences (P > 0.05) were observed between 12 and 18 h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P > 0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12 h, and the prolongation of that time for up to 18 h had no detrimental effect on fertilization. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Spermatozoa; Cleavage; Sperm selection; Sperm–oocyte co-incubation time
∗ Corresponding author. Tel.: +55-67-768-2150; fax: +55-67-768-2150. E-mail address: [email protected] (M.A.N. Dode).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 4 8 - 8
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1. Introduction Sperm selection methods are routinely applied on the in vitro fertilization (IVF) system for various species. These methods are used in order to remove seminal plasma/cryoprotectant and to increase sperm quality characteristics (Parrish et al., 1995; Rodriguez-Martinez et al., 1997). Several methods for sperm selection have been described, such as washing/centrifugation (Fukuda et al., 1990), density gradient centrifugation (Parrish et al., 1995), deferential filtration through a glass wool column (Stabbings and Wasik, 1991) and self-migration procedure (Lonergan et al., 1994). The most widely used of these methods are the swim-up and the Percoll centrifugation (Rodriguez-Martinez et al., 1997; Palomo et al., 1999), which are a self-migration and a density gradient procedure, respectively. The Percoll gradient provides a much higher recovery of motile spermatozoa compared to the swim-up (Parrish et al., 1995) and more successful IVF results. However, there is a variation among Percoll batches and some batches can be deleterious to spermatozoa affecting cleavage and blastocyst rates (Avery and Greve, 1995). On the other hand, the swim-up shows a lower recovery rate, but the sperm quality is variable compared to the Percoll method (Parrish et al., 1995; Correa and Zavos, 1996; Rheingantz et al., 2000). Sperm preparation for IVF can also be done by washing/centrifugation with no deleterious effect on cleavage or blastocyst rates (Avery and Greve, 1995). There are differences in the procedures of how each of those methods select motile from immotile spermatozoa. Centrifugation, used in both methods, can damage sperm with generation of reactive oxygen species (ROS) and peroxidation of sperm membranes, which can bring negative effects on sperm–oocyte fusion (Kim and Parthasarathy, 1998; Watson, 2000). In addition, it has been suggested that the passage of the spermatozoa through the Percoll gradient could induce capacitation (Schweitzer et al., 1996; Rosenkranz and Holzmann, 1997). Therefore, if the capacitation time is reduced in the Percoll gradient compared to the other methods, the co-incubation time could also be reduced avoiding the cytotoxic damage caused by the increased amount of reactive oxygen species (ROS) (Kim et al., 1999). In fact, a reduced exposure of oocyte to spermatozoa favors embryo viability in humans, possibly due to a decrease in potential damage from sperm metabolic waste products (Gianaroli et al., 1996; Dirnfeld et al., 1999). Furthermore, the earlier the spermatozoa are capacitated the earlier the penetration will occur avoiding the aging process of the oocyte, which can also cause abnormal fertilization. The present study was carried out to evaluate the effect of the sperm selection method on penetration and cleavage of Bos indicus in vitro matured oocytes. In addition, it was verified whether the sperm–oocyte co-incubation time affects fertilization. 2. Material and methods Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. Semen samples thawed in a water bath at 37 ◦ C were evaluated for motility and submitted to each one of the different treatments: Percoll, swim-up and washing. At the end of each treatment, the semen samples were used for IVF.
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2.1. Sperm selection procedures In the Percoll technique, an aliquot of 500 l of semen was layered on Percoll gradient consisting of 2 ml of 45 and 90% of Percoll and centrifuged at 700 g for 30 min. The Percoll 90% was prepared using 500 l of sp-Talp 10× in 4.5 ml of Percoll (Parrish and Eid, 1994). The 45% Percoll solution was prepared with 1 ml of 90% Percoll and 1 ml of sp-TALP. After centrifugation the supernant was discarded and the pellet was resuspended with 200 l of sp-TALP. In the swim-up procedure, semen sample was placed in a 15-ml centrifuge tube, in which 1 ml of sp-TALP (Parrish et al., 1998) was added, and incubated at 38.5 ◦ C for 1 h. After incubation, the upper 0.85 ml of the supernatant was collected and transferred to other tube and diluted with sp-TALP to give a final volume of 5 ml. Then, it was centrifuged at 120 g for 10 min. After centrifugation, the supernant was discarded and the pellet diluted with 200 l of sp-TALP. For the washing method, after thawing the semen sample was washed twice at 300 g for 5 min in sp-TALP. After the last centrifugation, the pellet was resuspended with 200 l of sp-TALP. 2.2. In vitro maturation (IVM) Ovaries from Nellore cows (Bos indicus) were collected just after slaughter and transported to the laboratory in saline solution (NaCl 0.9%) supplemented with penicillin G (100 IU/ml) and streptomycin sulfate (100 g/ml) at 30 ◦ C. Oocytes from 2 to 5 mm diameter follicles were aspirated with an 18-gauge needle and pooled in a 15-ml conical tube. After sedimentation, the cumulus oocyte complexes (COC) were recovered and selected by using a stereomicroscope. The good quality COCs were washed three times in maturation medium and transferred to a 200-l drop of maturation medium under paraffin oil and incubated for 24 h at 38.5 ◦ C in 5% of CO2 in air. The maturation medium consisted of TCM-199 supplemented with 10% FCS (v/v), 5.0 g/ml of LH (Sigma L-9773), 0.5 g/ml of FSH (Sigma F-8001) and antibiotics (100 IU/ml of penicillin and 100 g/ml of streptomycin), with 5% CO2 in air at 38.5 ◦ C. 2.3. In vitro fertilization (IVF) and embryo culture For IVF, COCs were washed and transferred to the 150-l drop of fertilization medium. Fertilization medium used was TALP (Parrish et al., 1998) supplemented with penicillamine (2 mM), hypotaurine (1 mM), epinephrine (250 mM) and heparin (10 g/ml). Motile spermatozoa obtained by the various sperm selection methods (Percoll gradient, swim-up and washing) were added into the fertilization drop in a final concentration of 1 × 106 spermatozoa/ml. Spermatozoa and oocytes were co-incubated for 3, 6, 12 and 18 h at 38.5 ◦ C with 5% of CO2 in air. After co-incubation, the zygotes were washed and transferred to the embryo culture medium. The medium was TCM-199, supplemented with 10% FCS (v/v), antibiotics (100 IU/ml of penicillin and100 g/ml of streptomycin) and oviductal cells. Oviduct epithelia cells were prepared on the day of ovary collection. The oviducts, transported
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to the laboratory in sterile saline solution at 4 ◦ C, were washed with PBS and dissected. To recover the cells the lumen contents were washed out of the oviduct down into a centrifuge tube. The cells recovered were washed 3 times by centrifugation with PBS (200 g/5 min) and once with culture medium. The final pellet was resuspended with culture medium and cultured for 1–2 days. At the preparation of the embryo culture, drop only cells with visible cilia movement were added. After different sperm–oocyte co-incubation time, the presumptive zygotes were cultured up to 44 h post insemination. The uncleaved oocytes were fixed and stained with lacmoid (1%) and examined under a phase contrast microscope to determine penetration, pronucleus formation and polyspermy. 2.4. Experimental design A total of 1002 in vitro, matured oocytes were used in a four-replicate experiment. After selection by each one of the methods used, sperm samples were evaluated and the concentration was determined. Semen was then added to the fertilization drop and was co-incubated with the oocytes for various periods of time. At 3, 6, 12 and 18 h post insemination, the oocytes were removed from the fertilization drop, washed and transfer into the embryo culture medium. Cleavage, penetration, pronucleus formation and polyspermy rates were determined 44 h post insemination. 2.5. Statistical analysis All data are expressed in percentage and were examined by analysis of variance, in which the effect of treatment, time of incubation, and interaction between treatments and time of incubation were evaluated. When a statistical significant effect was found the differences among treatments means were determined by least significant differences method at a probability value of 0.05% for all parameters. 3. Results The results of the ANOVA showed that there was no effect of sperm selection method in any of the variables studied. On the other hand, time of co-incubation had a significant effect (P < 0.05) in the percentage of penetration and cleavage. There was no interaction between method of selection and sperm–oocyte co-incubation time. Hence, the data for those variables were presented separately as independent variables. Data for penetration, pronucleus formation, polysperrmy and cleavage rates for each treatment at each time point are presented in Table 1. The comparison among the sperm selection methods showed no differences in the penetration or cleavage rate. Nor did any treatment have any significant effect on the formation of pronuclei, nor on the incidence of polyspermy (Table 2). The penetration rate at the time of the first testing (3 h) was low for all treatment (Table 3). The penetration rate increased from 6 to 12 h, but it remained the same for the following incubation time (12 and 18 h). Co-incubation time did not influence the pronucleus formation
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Table 1 Effect of sperm preparation treatment and time of co-incubation on IVF (means ± S.D.) Treatment
Number of oocytes
Time (h)
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
Swim-up
97 83 95 115 71 75 90 88 75 71 82 60
3 6 12 18 3 6 12 18 3 6 12 18
27.3 ± 4.5 68.4 ± 4.2 91.7 ± 1.0 93.4 ± 0.9 28.2 ± 2.3 68.2 ± 8.7 72.6 ± 5.7 74.3 ± 3.2 23.7 ± 2.7 53.3 ± 8.9 68.6 ± 5.4 69.6 ± 6.0
4.9 ± 1.7 3.1 ± 1.1 2.8 ± 1.3 3.9 ± 0.8 6.4 ± 1.8 2.3 ± 0.8 4.4 ± 0.9 0.9 ± 0.4 2.5 ± 1.3 5.4 ± 2.2 0.0 ± 0.0 2.0 ± 0.7
10.7 ± 1.7 10.6 ± 2.9 7.0 ± 1.4 2.0 ± 0.5 7.7 ± 1.6 4.2 ± 1.7 2.1 ± 0.6 4.9 ± 1.0 2.5 ± 0.8 0.0 ± 0.0 6.9 ± 2.1 3.0 ± 1.1
11.9 ± 1.9 53.8 ± 5.4 76.2 ± 2.8 84.8 ± 1.5 9.9 ± 1.9 61.6 ± 8.1 65.1 ± 4.9 69.2 ± 3.6 17.4 ± 0.9 47.9 ± 8.1 61.6 ± 6.0 61.1 ± 5.6
Percoll
Wash
Table 2 Effect of sperm selection method on fertilization characteristics and cleavage rates (means ± S.D. deviation)a Treatment
Number of oocytes
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
Swim-up Percoll Wash
390 324 288
70.3a ± 6.8 60.8a ± 6.7 53.8a ± 6.8
3.7a ± 1.3 3.5a ± 1.1 2.5a ± 1.2
7.6a ± 1.9 4.7a ± 1.1 3.1a ± 1.2
56.7a ± 7.2 51.5a ± 7.3 47.0a ± 6.5
a
Values with same letters are statistically similar (P > 0.05).
Table 3 Effect of sperm–oocyte co-incubation time on fertilization characteristics and cleavage rates (means ± S.D. deviation)a Time (h)
Number of oocytes
Oocytes penetrated (%)
Oocytes polyspermic (%)
Oocytes pronucleus (%)
Oocytes cleaved (%)
3 6 12 18
243 229 267 263
26.5a ± 3.8 63.3b ± 8.0 77.6b ± 5.7 79.1b ± 4.7
4.6a ± 1.8 3.6a ± 1.6 2.4a ± 1.2 2.3a ± 0.8
7.0a ± 1.8 4.9a ± 2.4 5.3a ± 1.7 3.3a ± 1.0
13.1a ± 1.9 54.4b ± 7.6 67.6c ± 5.5 71.8b ± 4.7
a
Values with different letters are statistically different (P > 0.05).
or the polyspermy rate. However, the percentage cleaved embryos gradually increase from 3 to 18 h of sperm–oocyte co-incubation time, although no statistical differences were detected between 12 and 18 h (Table 3). 4. Discussion With the advent of IVF and other assisted reproductive technologies, sperm selection became an important procedure to obtain optimal results. It is needed not only to
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remove seminal plasma or cryoprotector, but also to use the best spermatozoa available from a frozen/thawed semen samples. There are several sperm selection methods and the choice of method depends not only of the yield of motile spermatozoa, but also on its technical complexities, the material and equipment needed and the time costs (Mortimer, 1994). In the present study, we compared a very easy, fast and economic method, the washing with other more expensive and time consuming, the Percoll and the swim-up methods. Several reports indicated that the Percoll (Parrish et al., 1995; Correa and Zavos, 1996; Prakash et al., 1998) and swim-up (Risopatron et al., 1996; Palomo et al., 1999) method yield a qualitative superior sperm sample with significantly more motile, viable and morphologically normal spermatozoa than the washing procedure. In spite of the number of studies in this topic, none of these have verified the effect of the three methods simultaneously, on fertilization of bovine oocytes. In the present study, the different methods to select spermatozoa had similar effect on the in vitro penetration, pronucleus formation, polyspermy and cleavage rates. Similar results were obtained by Seidel et al. (1995) and Palomo et al. (1999), who compared the swim-up and Percoll methods. Risopatron et al. (1996), comparing the migration/sedimentation method and the washing procedure showed that the percentage of oocytes with male pronucleus formation was lower in the washing. However, Avery and Greve (1995) reported that the washing method can be used for preparation of sperm to IVF of bovine oocytes with no detrimental effect on cleavage or blastocyst rates. According to Larsson and Rodriguez-Matinez (2000), the cleavage rate is most suitable to evaluate semen samples than the blastocyst rates, most likely because blastocyst is much more dependent on culture conditions during embryo development than cleavage. Therefore, in the present study, we used cleavage as the end point and no differences were observed among treatments. These results indicate that, under the conditions used, a sufficient number of sperm capable of fertilizing were available, even if they had not been selected as occur in the washing method. Therefore, it is possible that the sperm concentration usually used for IVF can overcome the differences in the sperm quality obtained by different methods to select spermatozoa. It seems that in this case the three methods, studied can be used with no detrimental effect on fertilization. In humans, it has been suggested that short-time exposure of oocytes to spermatozoa improves IVF results (Gianaroli et al., 1996; Quinn et al., 1998; Dirnfeld et al., 1999), mainly because a long-time exposure of oocytes to a large number of spermatozoa would create suboptimal conditions due to excessive generation of ROS (Quinn et al., 1998). These products would induce peroxidation of the membrane lipids reducing membrane fluidity and impairing sperm function (Mortimer, 1994). Therefore, time of co-incubation is also an important aspect to be considered when one wants to increase IVF results. It has been reported that the movement of the spermatozoa in capacitation medium or its passage through the Percoll gradient could rinse out some surface glycoproteins and reduce capacitation time (Rosenkranz and Holzmann, 1997). Based in this assumption, it could be expected that the method used to prepare semen for IVF could alter the capacitation time and the life span of the spermatozoa differently, which could alter time required for fertilization. The results of the present study demonstrated that there was an effect of
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the co-incubation time on fertilization, but no interaction between time and sperm selection method occurred. Thus, the variation, which occurs among the various time periods, was similar for all methods. These results show that if the method influences capacitation, the change is not big enough to affect the co-incubation time required for fertilization. Penetration and cleavage rates were lower when the co-incubation time was 3 h which could be expected since spermatozoa needs at least 4 h to penetrate in vitro matured oocytes (Saeki et al., 1991; Pavlok, 2000). This was confirmed by the significant increase in both penetration and cleavage rates observed at 6 h of co-incubation. Fertilization results continued to increase up to 12 h of incubation demonstrating that regardless the method used there is heterogeneity of the sperm population and that spermatozoa do not complete capacitation simultaneously. Actually, even under the same conditions some spermatozoa from the same ejaculate capacitated faster than others (Yanagimachi, 1994). The incidence of polyspermy has been shown to increase as the time of co-incubation increases (Chian et al., 1992; Sumantri et al., 1997). However, a difference among bulls in terms of sperm capacitation times and acrosome reaction has also been reported (Parrish et al., 1986; Yanagimachi, 1994). In the present study, samples from the same bull were used, and no differences in polyspermy were observed for the various sperm–oocyte co-incubation times. The generation of ROS increases under in vitro conditions using 5% of CO2 in air (Kim et al., 1999), therefore as the time increases more of these products will be produced. In fact, in human, 16 h of incubation can be detrimental to IVF results (Quinn et al., 1998; Dirnfeld et al., 1999) due to excessive generation of ROS and other deleterious products from sperm metabolism into the IVF medium. Unlike in the humans, the incubation for 18 h had no detrimental effect on bovine IVF. It is possible that for the bovine those deleterious products are not high enough during that period to cause a significant effect on fertilization. These can be confirmed by the fact that addition of antioxidant during IVF in bovine (Kim et al., 1999) and porcine (Boquest et al., 1999) did not affected cleavage rates. In fact, although the ROS have many detrimental effects on cells they also have some beneficial effects, such as they are needed for acquisition of fertilizing ability by spermatozoa and hydrogen peroxide is required for induction of capacitation in some species (Blodin et al., 1997; Watson, 2000). Therefore, a delicate balance between the amount of ROS generated and scavenged determines whether the final outcome is detrimental or beneficial (Kim et al., 1999). In the present study, it seems that no alteration in these balance has occurred in 18 h of incubation, since cleavage rates were similar to the 12-h period. However, it is important to point out that there is a variation among bulls in regard to the production of ROS, with some bulls generating excessive amounts of ROS during IVF (Kim et al., 1999). It is possible that the bull used in this study does not produce high amounts of ROS avoiding all the problems caused by these products. In conclusion, it can be stated that under the conditions of these study Percoll, washing and swim-up can be used to prepare sperm for IVF with no detrimental effect on fertilization. In addition, regardless the method used better, fertilization results were obtained when sperm–oocyte were co-incubated for 12 h. However, the prolongation of that time for up to 18 h has no detrimental effect on fertilization.
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