Effect Of Sperm Treatment Research Articles

229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment. Supported by MEC (AGL2006-03495).

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

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

135 THE EFFECT OF DIFFERENT TREATMENTS OF PORCINE EJACULATED AND EPIDIDYMAL SPERMATOZOA ON ROS GENERATION

The production of pig embryos in vitro is still relatively inefficient compared with results obtained with oocytes matured and fertilized in vivo. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of embryos produced by IVM-IVF (Kikuchi et al. 2002 Biol. Reprod. 1033-1041). Between factors affecting polyspermy are the sperm procedures before IVF. Usually, these procedures including centrifugations that increase reactive oxygen species (ROS) formation in spermatozoa. ROS play an important role in sperm physiology including capacitation. Physiological concentrations of ROS have been proposed to enhance sperm capacitation by increasing cAMP synthesis and by inhibiting protein tyrosine phosphatases whilst activating tyrosine kinases. In general, epididymal spermatozoa appear to be able to capacitate and fertilize eggs in vitro much more easily than ejaculated spermatozoa (Yanagimachi Mammalian Fertilization. In: The Physiology of Reproduction, Raven Press 1988; 135-182). In this study, we investigated how different sources (ejaculated spermatozoa vs. epididymal spermatozoa) and sperm capacitating methods, usually employed in porcine IVF, could be affect ROS generation. Sperm-rich fractions from five fertile boars and sperm from five different epididyimides were used. The semen samples were then: (i) washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA, (ii) left unwashed, or (iii) washed on a Percoll (Pharmacia, Uppsala, Sweden) gradient (Mat�s et al. 2003 Reproduction 125, 133-141). Production of ROS was measured by incubating the spermatozoa in the in vitro fertilization medium (TALP) in the presence of 0.7 �m 22,72-dichlorodihydrofluorescein diacetate at 37�C under 5% CO2 in 100% humidified air. Every 15 min (from 15 to 135) the samples were analyzed and evaluated by flow cytometry. Measurements were expressed as the mean green intensity fluorescence units and it was used as index of ROS generation (Gadea et al. 2005 J. Androl. 26, 396-404). ANOVA analysis revealed a significant effect of sperm treatment on the ROS generation (P < 0.001). The highest value was obtained in sperm washed on a Percoll gradient and the lowest in unwashed semen. When ejaculated vs. epididymal semen was analyzed, the same tendency was observed in both. However, the values were always lower in epididymal semen than in ejaculated semen (P < 0.001). As a conclusion, ROS generation is different between treatments and between semen procedures for the time interval studied, and this finding may help to explain the different outcome in IVF among laboratories. This work was supported by Ministerio de Educaci�n y Ciencia, AGL2003-03144.