Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.
BACKGROUND
In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding.


RESULTS
Experiment 1, 4-20 replicates with total 821in vitromatured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents (P< 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates;n= 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively (P< 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) (P> 0.05).


CONCLUSIONS
Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.
División de Ciencias Biológicas y de la Salud, Departamento de Producción Agrícola y Animal, Universidad Autónoma Metropolitana-Xochimilco, Ciudad de México, Mexico ; Doctorado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico.
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Hernández-Pichardo
División de Ciencias Biológicas y de la Salud, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, CP 09340 Ciudad de México, Mexico.
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Ducolomb
Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores Cuautitlán, UNAM, Ciudad de México, Estado de México Mexico.
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Romo
Conservation, Genetics & Biotech, LLC, Valley City, ND USA.
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Kjelland
División de Ciencias Biológicas y de la Salud, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, CP 09340 Ciudad de México, Mexico.
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Fierro
División de Ciencias Biológicas y de la Salud, Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico.
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Casillas
División de Ciencias Biológicas y de la Salud, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, CP 09340 Ciudad de México, Mexico.
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Betancourt
Ethanol; ICSI; Oocyte activation; Ovine; Pronucleus; Zona pellucida