Snider Alexandria P, Workman Aspen M, Heaton Michael P, Vander Ley Brian L, Krueger Alexandria C, Sonstegard Tad S
US Meat Animal Research Center, USDA, Agricultural Research Service, 844 Road 313, Spur 18 D, Clay Center, NE 68933, USA.
Great Plains Veterinary Educational Center, University of Nebraska-Lincoln, 820 Road 313 Spur 18 D, Clay Center, Nebraska 68933, USA.
Biol Reprod. 2025 Feb 14;112(2):245-252. doi: 10.1093/biolre/ioae169.
Bovine viral diarrhea virus (BVDV) infection during pregnancy is a significant contributor to reproductive failures in cattle. The bovine receptor for BVDV (CD46) was previously edited with a six amino acid substitution (G82QVLAL to A82LPTFS) and shown to have significantly reduced BVDV susceptibility in a Gir heifer calf. Since a role for CD46 has been proposed in mammalian fertilization, our objective was to assess the edited heifer's fertilization rates, early embryonic development, and germline transmission conformation of the edit. Cumulus oocyte complexes were collected from the edited heifer and unedited females, fertilized with semen from an unedited bull and cultured until the blastocyst stage. Ultrasound examinations and serum progesterone concentration were also monitored to confirm estrous cyclicity in the CD46-edited heifer. Estrous cyclicity was normal with visualization of a corpus luteum and elevated progesterone concentrations. Fertilization rates and blastocyst development were not different in oocytes from edited and unedited controls. Genome sequence analysis of blastocysts confirmed germline transmission of either edited allele from the heifer. Subsequently, the CD46-edited heifer was artificially inseminated with semen from an unedited Gir bull and fertility status was confirmed with a diagnosed conception at Day 35 of gestation. Thus, a six amino acid substitution in CD46 did not negatively affect fertilization of edited oocytes or early embryonic development when fertilized with semen from an unedited bull. An edited bull is still needed to similarly evaluate reproductive function of sperm cells carrying this CD46 edit.
孕期感染牛病毒性腹泻病毒（BVDV）是导致奶牛繁殖失败的一个重要因素。先前已对牛BVDV受体（CD46）进行编辑，使其发生六个氨基酸的替换（从G82QVLAL变为A82LPTFS），并且在一头吉尔小母牛犊中显示出对BVDV的易感性显著降低。由于已有人提出CD46在哺乳动物受精过程中发挥作用，我们的目标是评估经编辑的小母牛的受精率、早期胚胎发育以及该编辑的种系传递情况。从经编辑的小母牛和未经编辑的母牛采集卵丘卵母细胞复合体，用未经编辑的公牛的精液进行受精，并培养至囊胚阶段。还通过超声检查和监测血清孕酮浓度来确认经CD46编辑的小母牛的发情周期。通过观察黄体和升高的孕酮浓度证实发情周期正常。经编辑和未经编辑的对照的卵母细胞的受精率和囊胚发育没有差异。对囊胚进行基因组序列分析证实了来自小母牛的经编辑等位基因的种系传递。随后，用未经编辑的吉尔公牛的精液对经CD46编辑的小母牛进行人工授精，并在妊娠第35天通过确诊妊娠来确认生育状态。因此，当用未经编辑的公牛的精液受精时，CD46中的六个氨基酸替换不会对经编辑的卵母细胞的受精或早期胚胎发育产生负面影响。仍需要一头经编辑的公牛来类似地评估携带这种CD46编辑的精子细胞的生殖功能。
