Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

Abstract

Abstract. This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa. Key words: Epididymal spermatozoa, In vitro fertilization (IVF), Mouse, Piezo-assisted micromanipulation, Vitrification (J. Reprod. Dev. 54: [107][108][109][110][111][112] 2008) he epididymis is an essential organ for sperm development and storage. The epithelial cells of the epididymis can secrete certain proteins to promote maturation of spermatozoa [1][2][3], and the specific environment within the epididymis, characterized by a low pH and hyperosmotic pressure, allows spermatozoa to survive for several weeks [4,5]. Spermatozoa retrieved from the epididymides of dead animals retain the ability to penetrate and fertilize female gametes and can be used in genetic preservation of endangered male animals. Recently, some studies on preserving the epididymis at refrigerator temperatures in order to retrieve spermatozoa for in vitro fertilization (IVF) or artificial insemination have been carried out in deer [6][7][8] [12]. Research on storage of the mouse epididymis is important since the mouse is most commonly used in biomedical research. Shipping the refrigerated epididymides (spermatozoa) of mouse strains avoids th...

DNA methylation pattern in mouse oocytes and theirin vitrofertilized early embryos: effect of oocyte vitrification

“…Despite recent technological advances, cryopreservation of mammalian oocytes remains difficult, as reflected by reduced rates of fertilization and embryonic development compared with those of fresh oocytes and embryos (Chen et al ., 2001; Larman et al ., 2006; Tian et al ., 2007; Fan et al ., 2008; Li et al ., 2011). In order to improve the efficiency of oocyte cryopreservation, the OPS method for oocyte vitrification was developed subsequently (Vajta et al ., 1998).…”

Successful pregnancy after intracytoplasmic sperm injection with testicular spermatozoa transported only under refrigeration

“…Several studies have reported the preservation of epididymides by using mineral oil under refrigeration [21,22]. We considered that placing the seminiferous tubules in mineral oil would make them resistant to temperature change.…”

“…Several studies on the preservation of sperm under refrigeration have reported optimum temperature control of 4-6°C [11,12,[22][23][24][25]. Furthermore, one study reported that the transportation of C57BL/6 mouse strain epididymides was more successful at 7°C than at 5°C [21].…”

“…Furthermore, one study reported that the transportation of C57BL/6 mouse strain epididymides was more successful at 7°C than at 5°C [21]. We decided on a temperature control of 6°C because taking the findings of these studies [11,12,[22][23][24][25] into consideration offered the best chance for success.…”

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

“…The low pH and hyperosmotic pressure within the epididymis allow spermatozoa to survive for several days at body temperature (Acott & Carr, 1984; De Pauw et al ., 2003). Recently, some studies on preserving the epididymis at refrigerator temperature (4 °C), to retrieve spermatozoa for in vitro fertilization or artificial insemination, have been carried out in the mouse (Fan et al ., 2008), deer (Hishinuma et al ., 2003; Soler et al ., 2003; Martinez-Pastor et al ., 2005), pigs (Kikuchi K, 1999), dogs (Yu & Leibo, 2002) and sheep (Kaabi et al ., 2003). These results suggest that spermatozoa held within the epididymis are more tolerant for preservation at room temperature than are isolated spermatozoa, but, to our knowledge, there is no report on room temperature preservation of sperm in the epididymis.…”