Chot Onpreeya, Thongkham Marninphan, Satsook Apinya, Arjin Chaiwat, Mekchay Supamit, Hongsibsong Surat, Sringarm Korawan
Livestock innovative and biocirculation laboratory, Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand; Center of Excellence on Agricultural Biotechnology, Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation, Thailand (AG-BIO/MHESI), Bangkok, Thailand.
Livestock innovative and biocirculation laboratory, Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand.
Reprod Biol. 2025 Jun;25(2):101009. doi: 10.1016/j.repbio.2025.101009. Epub 2025 Mar 15.
The pig industry is interested in increasing the number of female piglets by using sexed semen. Immunological sperm sexing is a promising method. This study investigated and produced a monoclonal antibody (MAbs) targeted to plasma membrane epitopes on porcine Y-chromosome-bearing sperm. Two BALB/c mice were immunized with 92.08 % high-purity porcine Y-sperm, which was separated by a cell sorter flow cytometer. The hybridoma cells were a fusion of myeloma cells (P3X63Ag8.653) and splenocyte cells from immunized mice. Indirect ELISA screening for positive antibodies produced by a single clone well (C2B2) with a high titer specific to porcine Y-sperm. The C2B2 clone was used to produce and purify C2B2-MAbs, yielding 2.78 ± 0.78 µg/mL. The C2B2-MAbs was highly specific to Y-sperm (100.00 %) and had a low cross-reactivity with X-sperm (3.25 %). Therefore, the percentage cross-reactivity of C2B2-MAbs was low for conventional sperm from various livestock, including 0.34 % for Angus, 0.38 % for Holstein-Friesian, 0.20 % for goats, and 0.25 % for buffalo. The bright fluorescence of FITC displayed by the C2B2-MAbs bound to the plasma membrane of porcine Y-sperm provided evidence of affinity between them. However, the C2B2-MAbs bound to an X-sperm lacked fluorescence. C2B2-MAbs showed specificity for the plasma membrane of porcine Y-sperm, which can be used in porcine semen sexing in further studies.
养猪业对通过使用性别分选精液来增加雌性仔猪数量很感兴趣。免疫精子性别分选是一种很有前景的方法。本研究调查并制备了一种针对携带猪Y染色体精子质膜表位的单克隆抗体（MAbs）。用细胞分选流式细胞仪分离得到的纯度为92.08%的高纯度猪Y精子免疫两只BALB/c小鼠。杂交瘤细胞是骨髓瘤细胞（P3X63Ag8.653）与免疫小鼠的脾细胞融合而成。通过间接ELISA筛选由单克隆孔（C2B2）产生的对猪Y精子具有高滴度特异性的阳性抗体。使用C2B2克隆来生产和纯化C2B2 - MAbs，产量为2.78±0.78μg/mL。C2B2 - MAbs对Y精子具有高度特异性（100.00%），与X精子的交叉反应性较低（3.25%）。因此，C2B2 - MAbs对包括安格斯牛0.34%、荷斯坦 - 弗里生牛0.38%、山羊0.20%和水牛0.25%在内的各种家畜的常规精子的交叉反应率较低。与猪Y精子质膜结合的C2B2 - MAbs所显示的FITC明亮荧光证明了它们之间的亲和力。然而，与X精子结合的C2B2 - MAbs没有荧光。C2B2 - MAbs对猪Y精子的质膜具有特异性，可用于进一步研究中的猪精液性别分选。
