Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

J.E.Hernández-Pichardo; Y.Ducolomb; S.Romo; M.E.Kjelland; R.Fierro; F.Casillas; M.Betancourt

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Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

机译：使用化学活化的绵羊卵母细胞和透明带结合的精子通过ICSI形成前核

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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 821 in vitro matured 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 ICSl (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.

机译：背景：为了改善ICSI，必须进行适当的精子选择和卵母细胞活化。本研究的目的是确定使用ICSI与化学活化的绵羊卵母细胞和通过向上游泳（SU）或向上游泳选择的精子进行受精的效率。结果：实验1,4-20复制，共821个体外成熟卵母细胞用乙醇，钙离子载体或离子霉素化学激活，以确定卵母细胞激活（精确到1个PN）。结果（分别为54,47,42％），但与假，ICSI和假注射中的机械活化卵母细胞（分别为13,25,32％）相比，机械激活的卵母细胞（分别为13,25,32％）有统计学差异（P ＜0.05）（10-17个重复; n = 429）实验2：使用十二个射精和28个精液吸管（11-19个重复），通过SU在BSA-TCM 199-H培养基中选择精子，共分析了2,294个新鲜精子和冷冻融化后的精液中的2,760个精子SU或SU + ZP结合SU所选择的新鲜精子wd顶体反应（AR）为59％，通过SU + ZP结合选择的精子的AR增至91％。相比之下，使用SU或SU + ZP结合的冻融精子的AR分别为77和86％（P ＜0.05）。实验3：200个机械活化卵母细胞受精（17个重复）为4％，但ICS1后乙醇活化卵母细胞受精（12-28％）（5-6个重复）增加。当新鲜精子仅由SU选择时123个卵母细胞中，受精率达到28％; SU + ZP选择的精子中卵母细胞为25％（73个卵母细胞）。相比之下，SU选择的冻融精子中，卵子中受精率为13％（70个卵母细胞）。），而SU + ZP结合产生的精子占12％（51个卵母细胞）（P＞ 0.05）。结论：化学激活比机械激活诱导更高的卵母细胞激活。与SU和ZP相比，SU + ZP在新鲜和冷冻食品中提高了AR / A和AR / D率因此，就受精率而言，ICSI后的化学活化与机械活化相比，ICSI后卵母PN形成的化学活化增加。此外，SU和SU + ZP处理的新鲜精子与冻融的精子有显着差异，但是精子处理没有显着差异。

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《畜牧与生物技术杂志(英文版)》 |2017年第2期|370-377|共8页
作者
J.E.Hernández-Pichardo; Y.Ducolomb; S.Romo; M.E.Kjelland; R.Fierro; F.Casillas; M.Betancourt;
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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;

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;

Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores Cuautitlán, UNAM, Ciudad de México, Estado de México,Mexico;

Conservation, Genetics & Biotech, LLC, Valley City, ND, USA;

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;

División de Ciencias Biológicas y de la Salud, Departamento de Biología de la Reproducción, Universidad Autcónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico;

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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Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

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