An efficient system to collect large numbers of vital zygotes is a pre-requisite for application of zygote genome-editing technology, including development of efficient models for xenotransplantation using pigs. Owing to the sub-optimal in vitro production of zygotes in pigs, efficient collection of in vivo developed zygotes is required. Timing of ovulation is a key factor to sustain efficiency since the interval between pronuclear formation and the first division is very short in pigs. The weaning-to-estrus interval can, due to its inverse relation with length of estrus and time of ovulation, interfere with ovulation and make it asynchronous, which reduces the probability of obtaining zygotes. This retrospective study compared the effects of three weaning-to-estrus intervals of 3, 4 or 5 days on zygote collection efficiency in a total of 17 trials over a 3-year period including 223 sows. Donor sows in groups of 10-15 animals were super-ovulated with eCG 24 h after weaning and those in estrus at 48-72 h post-eCG were immediately treated with hCG, followed by insemination 6 and 24 h thereafter. Collected structures during laparotomy on Day 2 (Day 0: onset of estrus) were morphologically evaluated and only those with a single cell and two visible polar bodies were considered as zygotes. Zygotes were injected with CRISPR-Cas9 editor mixture and cultured for 6 days to evaluate their developmental ability against non-injected control zygotes. Of all recovered structures (N = 5,468), 67.4%, 30.8% and 1.8% were zygotes, 2-cell embryos and oocytes-degenerated embryos, respectively. The different weaning-to-estrus intervals did not affect either the percentages of collected zygotes (range: 64.1%-70.0%) or the percentages of sows with zygotes at collection time (range: 69.0%-73.3%). The weaning-to-estrus intervals did not affect the in vitro developmental ability of zygotes. After 24 h of culture, 78.1 ± 2.0% and 95.1 ± 0.6 (P < 0.05) of injected (N = 2,345) and non-injected (N = 335) zygotes, respectively, developed to 2-to-4-cell embryo stage. The total efficiency of the system was 64.1 ± 2.2% and 85.8 ± 1.5% (P < 0.05) for injected and non-injected zygotes, respectively. In conclusion, the results indicate that neither the efficiency of collecting in vivo derived porcine zygotes from superovulated sows nor the zygote ability to develop to blastocyst after cytoplasmic genome-editing injection were affected by a weaning-to-estrus interval between 3-to-5 days.
Keywords: CRISPR-Cas9; Cytoplasmic injection; Embryo collection; Pig; Weaned to estrus interval; Zygote.
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