Bragança G M, Alcântara-Neto A S, Batista R I T P, Brandão F Z, Freitas V J F, Mermillod P, Souza-Fabjan J M G
INRA, UMR7247, Physiology and Control de Reproduction et des Comportements, INRA, CNRS, Nouzilly, France; Faculdade de Veterinária, Universidade Federal Fluminense, Rua Vital Brazil, 64, CEP 24320-340, Niterói, RJ, Brazil.
INRA, UMR7247, Physiology and Control de Reproduction et des Comportements, INRA, CNRS, Nouzilly, France.
Theriogenology. 2021 Jul 1;168:59-65. doi: 10.1016/j.theriogenology.2021.03.022. Epub 2021 Apr 1.
The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 10 cells/mL) for 18 h in SOF medium supplemented with 5 μg/mL of heparin, 4 μg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x10 sperm per mL is used.
i)已知参与发情期山羊输卵管液(OF)早期繁殖的蛋白质(输卵管特异性糖蛋白,OVGP1;热休克蛋白-70A,HSPA1A;热休克蛋白-A8,HSPA8;膜联蛋白A1,ANXA1;膜联蛋白A5,ANXA5;以及肌球蛋白-9,MYH9)的存在;ii)体外受精期间其对多精受精调节和胚胎发育的功能影响。将体外成熟的卵母细胞与精子(1.0、2.0或4.0×10⁶个细胞/mL)在补充有5μg/mL肝素、4μg/mL庆大霉素和10%发情期绵羊血清的SOF培养基中共同培养18小时(CTRL1、CTRL2和CTRL4组),或在相同培养基中加入从发情期山羊获得的10% OF(OF1、OF2和OF4组)。通过蛋白质印迹法对OF进行分析,证实了所检测的六种蛋白质的存在。精子浓度的增加对任何组的穿透率均无影响(P>0.05);然而,在OF组和CTRL组中,随着精子浓度的增加,单精受精率均下降。无论使用何种浓度,汇总数据时,添加OF可提高(P<0.05)单精受精率,并倾向于(P=0.057)提高体外受精效率。此外,OF1组的体外受精效率高于OF4组(P<0.05)[60±13%对37±5%]。精子浓度和OF处理对发育能力没有影响(P>0.05),平均值为卵裂(72±2.6%)、囊胚(37±3.0%)、囊胚与卵裂的比例(51±4.8%)、孵化(62±1.2%)以及每个囊胚的细胞数(174±1.8)。总之,所分析的六种蛋白质存在于发情期山羊的OF中,体外受精期间添加该OF可能调节多精受精发生率并提高体外受精效率,尤其是当每毫升使用1×10⁶个精子时。