PDF《In vitro Fertilization》

2004 and

2023 as positive control. Freshly ejaculated semen was immediately diluted in Beltsville thawing solution and sperm motility was measured. Spermatozoa with an initial motility of more than 75% were used in further analysis. Evaluation of sperm parameters A

20 μL aliquot spermatozoa was placed into a pre- warmed slide glass and analyzed by computer assisted semen analysis (CASA, Hamilton Thorne, Beverly, MA, USA) system. The parameters recorded for each sample were as follows: percentage of motile sperm, movement velocity such as rapid, medium, slow, and static, and movement characteristics such as progressiveness, average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), sperm head elongation, and area rate. Acrosome staining assay by fluorescein isothiocyanate- Pisum sativum agglutinin Aliquots of boar sperm suspensions were smeared on glass slides and air-dried. Slides were fixed in 100% methanol for

10 min at room temperature. Thereafter, the smears of boar sperm suspensions were incubated with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC- PSA) (40 μg/mL phosphate buffered saline [PBS], pH 7.4, Sigma-Aldrich, St Louis, MO, USA;

L-0770) solution for

30 min at room temperature in a dark moisture chamber. The unbound FITC-PSA solution was washed out

5 times by PBS. Finally, a mounting solution (DAKO, Carpinteria, CA, USA;

S3025) was used to fix the boar sperm smears. In vitro fertilization and culture Semen samples from each boar were divided to make at least

10 aliquots and each aliquots was washed two times by centrifugation with Dulbecco'

s phosphate buffered saline (DPBS) supplemented with 0.1% bovine serum albumin at 2,000 g for

2 min. After washing, the sperm pellet was resuspended in modified Tris-buffered medium (mTBM) (Abeydeera and Day, 1997), which was pre-equilibrated for

18 h at 39°C under 5% CO2. Just before fertilization, the sperm motility from each aliquot was assessed and sperm Table 1. Breeding performance of semen from boar number 2004, 2012, and

2013 Boar number Age (Month) No. of sow for AI No. of pregnant sow (Pregnant rate, %) Delivery rate (%) Average No. of farrow Average No. of suckling piglet

2004 30

200 143 (71.5)

51 11.0 10.2

2012 16

159 79 (49.7)

50 9.2 8.3

2023 16

201 141 (70.1)

50 12.3 10.9 AI, artificial insemination. Lee et al. (2014) Asian Australas. J. Anim. Sci. 27:1417-1425

1419 with more than 80% motility were used for in vitro fertilization (IVF). After

40 to

42 h of in vitro maturation of porcine oocytes, the cumulus cells were denuded by gentle pipetting with 0.1% hyaluronidase and washed three times in Tyrode'

s medium containing 0.05% (w/v) polyvinyl alcohol (Bavister et al., 1983). Oocytes with even ooplasm and visible first polar body were used for all experiments. Groups of

15 ........