Baldassarre Hernan, Currin Luke, Michalovic Laura, Bellefleur Anne-Marie, Gutierrez Karina, Mondadori Rafael G, Glanzner Werner G, Schuermann Yasmin, Bohrer Rodrigo C, Dicks Naomi, Lopez Rosalba, Grand François-Xavier, Vigneault Christian, Blondin Patrick, Gourdon Jim, Bordignon Vilceu
Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada.
Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada.
Theriogenology. 2018 Aug;116:64-70. doi: 10.1016/j.theriogenology.2018.05.005. Epub 2018 May 26.
Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ± 16 vs. 38.5 ± 25 vs. 31.1 ± 22), mean oocytes recovered (26.5 ± 14 vs. 21.6 ± 10 vs. 19.4 ± 14) and cleavage rate (66.0 ± 14 vs. 61.1 ± 11 vs. 72.2 ± 8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5 ± 8%) compared with FSH12h (8.9 ± 5%, P < 0.05). Oocytes from follicles of ≥5 mm in diameter yielded a higher rate (P < 0.05) of development to the blastocyst stage (13.8%) than those collected from <5 mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 and > 130 days of age, did not affect the mean number of follicles (34.2 ± 15 vs. 39.3 ± 26 vs. 31.6 ± 25), the mean number of oocytes recovered (21.2 ± 10 vs. 24.5 ± 15 vs. 22.6 ± 17), and the cleavage rate (68.6 ± 11 vs. 61.7 ± 12 vs. 70.7 ± 10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9 ± 6 vs. 9.5 ± 8%, P < 0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1 ± 47 vs. 91.5 ± 25, P < 0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50 μM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1 ± 8 vs. 18.6 ± 4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.
对犊牛进行腹腔镜采卵（LOPU），随后进行体外胚胎生产（IVEP）并将胚胎移植（ET）到成年受体母牛体内，通过缩短世代间隔，在加速遗传进展方面具有巨大潜力。在本研究中，11头荷斯坦犊牛在2至6月龄期间，每隔2 - 3周接受多达6次LOPU操作。所有情况下，动物在LOPU前5天接受阴道孕酮释放装置（CIDR），并在LOPU前72小时开始使用三种方案之一进行促性腺激素刺激，在研究期间这三种方案在动物间轮换使用两次。犊牛每隔12小时（FSH12h）、或每隔8小时（FSH8h）注射促卵泡素（FSH），或每隔8小时注射FSH直至距LOPU前36小时，此时用单剂量400国际单位的孕马血清促性腺激素（eCG）替代FSH（FSH8h - eCG）。在可用于抽吸的平均卵泡数（分别为35.7±16、38.5±25、31.1±22）、回收的平均卵母细胞数（分别为26.5±14、21.6±10、19.4±14）和卵裂率（分别为66.0±14、61.1±11、72.2±8）方面，FSH12h、FSH捌h和FSH8h - eCG这三种处理之间未观察到统计学差异。然而，与FSH12h（8.9±5%）相比，FSH8h - eCG产生的可移植胚胎率显著更高（17.5±8%，P<0.05）。直径≥5毫米卵泡中的卵母细胞发育至囊胚阶段的比率（13.8%）高于直径<5毫米卵泡中采集的卵母细胞（6.8%），差异有统计学意义（P<0.05）。通过比较年龄<100天、101至130天和>130天的动物，动物年龄并不影响平均卵泡数（分别为34.2±15、39.3±26、31.6±25）、回收的平均卵母细胞数（分别为21.2±10、24.5±15、22.6±17）和卵裂率（分别为68.6±11、61.7±12、70.7±10%）。然而，与年龄较小的动物相比，年龄较大组的动物发育至囊胚阶段的比率显著更高（19.9±6对9.5±8%，P<0.01），且胚胎质量更好，表现为平均细胞数更高（119.1±4与91.5±25，P<0.05）。最后，我们通过在培养基中添加50μM牛磺熊去氧胆酸（TUDCA）来测试缓解内质网应激的益处，发现与对照培养相比，发育至囊胚阶段的比率在数值上更高（21.1±8对18.6±4%），但无统计学差异。总体而言，我们的研究结果表明，荷斯坦犊牛在6月龄前可产生大量可移植胚胎（范围为10 - 30个）。
