Moss C et al. (2024), A double ovulation protocol for Xenopus laevis ...

XB-ART-60810

PLoS One 2024 Jan 01;197:e0299179. doi: 10.1371/journal.pone.0299179.

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A double ovulation protocol for Xenopus laevis produces doubled fertilisation yield and moderately transiently elevated corticosterone levels without loss of egg quality.

Moss C , Vacca B , Arnold J , Hubens C , Lynch DM , Pegge J , Green MAR , Hosie CA , Smith TE , Green JBA .

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The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate oogenesis, fertilisation, and development at embryonic, larval, and metamorphic stages. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. After being rested for a minimum of three months, animals are re-used. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate as a measure of stress response for the two protocol groups over seven 3-month cycles. We found no differences in egg number-per-ovulation or egg quality between the groups and no long-term changes in any measures over the 21-month trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment) in the latter. However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within the levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research.

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Species referenced: Xenopus laevis
GO keywords: fertilization [+]

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	Fig 1. X. laevis egg yields and fertilisation rates under single- and double-ovulation protocols are similar. A, E, B, F: Numbers of eggs laid in 10 hours (9.00 am to 7.00 pm following hCG injection the previous evening) for each condition in the specific ovulation cycles shown (A, E) and for all four ovulation cycles pooled (B, F). C, G, D, H: Percentage of fertilised (cleaving) eggs from a ~200 egg sample in the specific ovulation cycles shown (C, G) and for all four ovulation cycles pooled (D, H). Data are plotted as box-and-whisker format (whiskers at 10/90th percentiles, bar at median, cross at mean) to show variability (A-D) and re-plotted as histograms (mean +/- standard deviation) (E-H) to show trends at a glance. Sample numbers: n ≥ 31 for each condition in each cycle and so ≥ 124 for pooled cycles (see Supporting Information S1 Appendix for raw data). https://doi.org/10.1371/journal.pone.0299179.g001
	Table 1. Summary of female corticosterone secretion rates.
	Fig 2. X. laevis corticosterone secretion under single- and double-ovulation protocols. Corticosterone secretion (pg/hr) values for each condition in the specific ovulation cycles shown (panels A and C) and for all four ovulation cycles pooled (panels B and D). Data are plotted as box-and-whisker format (whiskers at 10/90th percentiles, bar at median, cross at mean) to show variability (panels A and C) and re-plotted as histograms (mean +/- standard deviation) (panels B and D) to show trends at a glance. https://doi.org/10.1371/journal.pone.0299179.g002
	Fig 3. X. laevis female weights increased similarly under single- and double-ovulation protocols. Ratios of weights at the beginning and end of the trial (21 months) as plotted showing mean +/- standard deviation showing no significant difference between the groups (two-tailed paired t-test. https://doi.org/10.1371/journal.pone.0299179.g003

References [+] :

Bellerby, The endocrine factors concerned in the control of the ovarian cycle: Xenopus laevis as a test animal. 1933, Pubmed, Xenbase