The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

Abstract. Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind. Key words: Canine, Cryopreservation, Skim milk/glucose-based extender, Spermatozoa (J. Reprod. Dev. 54: [290][291][292][293][294] 2008) lthough freezing of canine semen and insemination of canine bitches with frozen-thawed semen is not as commonly used as in bovine and equine animals, successful artificial insemination with frozen canine semen has been well documented [1] since the first conception in 1969 [2]. Cryopreservation of canine spermatozoa offers potential exchange of genetic material, and thus may lead to improvement in the breeding management programs used to produce working dogs. In particular, in guide dog colonies, application of transcervical artificial insemination using frozen canine semen is anticipated to assist with meeting the demand for adequate supply of guide dogs for the blind. Egg yolk is the most commonly used compound in canine semen extenders for protection of spermatozoa from cold shock and disruption during the freezing and thawing process [1]. However, due to a recent outbreak of avian influenza and its triggering of growing concern throughout the world, transportation of frozen or chilled semen exposed to egg yolk has become extremely difficult. Several countries have, in fact, prohibited export and import of canine frozen semen that contains egg yolk. Thus, it is an urgent matter to develop a novel semen extender without egg yolk for use in freezing of canine spermatozoa. As an alterative compound to egg yolk, skim milk seems to be especially suitable as a semen extender in canine species, since a skim milk extender is the most commonly used extender for mouse [3] and goat [4] sperm. We report here successful artificial insemination with canine spermatozoa frozen in a solution containing skim milk, glucose and glycerol. Materials and Methods Colle...

In porcine in vitro fertilization (IVF), polyspermy is a persistent obstacle to the efficient production of normal embryos. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) was developed to isolate motile human spermatozoa (from diluted semen by two laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. We previously reported that the monospermy rate was higher when oocytes were co-cultured with isolated spermatozoa in an MFSS for 5 min than when spermatozoa were co-cultured traditionally in drops for 8 h (P < 0.05; Reprod. Fertil. Dev. 20, 187 abst). The present study was undertaken to compare the effect of oocyte location within the MFSS chamber on early development after IVF in the MFSS. A sperm-rich fraction from Berkshire boars was diluted at 1 × 108 cells mL–1 with modified Modena solution containing 20% seminal fluid. In the first experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine (mm199-caf) for 5 min at room temperature. Before flowing, porcine IVM oocytes were positioned in a part of the MFSS chamber where motile spermatozoa would accumulate with mm199-caf. After flowing for 5 min, those oocytes were cultured in caffeine-free mm199 for 8 h and then in a chemically defined medium (PZM-5) for 7 days. In the second experiment, denuded oocytes were co-cultured with isolated spermatozoa at several locations in the MFSS chamber, and the penetration and monospermy rates were estimated. The concentration of motile spermatozoa was also measured at each place in the MFSS chamber after isolation for 5 min. Statistical analyses of results based on 4 to 5 replicates were carried out by ANOVA and Fisher’s PLSD post hoc test (significance, P < 0.05). When IVM oocytes were co-cultured with spermatozoa gradually accumulated in the chamber of the MFSS for 5 min, the cleavage rate (83.7 ± 6.3% of 121 oocytes) was not different from that of control oocytes co-cultured with spermatozoa (5.7 × 105 cells mL–1) in 100-μL drops for 5 min (84.6 ± 6.6% of 126 oocytes). However, the blastocyst formation rate (38.2 ± 3.3%) was higher than for the controls (20.6 ± 6.8%; P < 0.05). After flowing for 5 min, the distance from the inflow opening of the MFSS chamber to the location of the oocytes did not affect the sperm penetration rate, but did affect the monospermy rate (14.0 ± 4.0% of 48 oocytes at the nearest position to 50.0 ± 5.6% of 43 oocytes at the furthest position; P < 0.05). After flowing for 5 min, the concentration of motile spermatozoa was also different at each location (57.5 ± 5.6 × 104 cells mL–1 at the nearest position to 0.8 ± 0.5 × 104 cells mL–1 at the furthest position; P < 0.05). These observations demonstrate that co-culturing oocytes with spermatozoa that gradually accumulated in the MFSS chamber improved the efficiency of blastocyst formation in the pig, whereas efficiency was affected by the position where oocytes were located in the chamber.

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.

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