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T. R. Sanford, M. De and G. W. Wood

Summary. Morphological and immunohistochemical analyses have documented the development of an acute inflammatory response, marked by the early appearance of granulocytes and later infiltration of mononuclear cells, in the uterus immediately after mating in mice. The response peaked on Day 1 and subsided by Day 3. In the present study, RNAs for macrophage colony-stimulating factor (CSF-1) and granulocyte–macrophage colony-stimulating factor (GM-CSF) and for interleukin lα (IL-1α), IL-1 β, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) were detected in uterine tissue on Day 1. With the exception of IL-6, which was higher on Day 3 than on Day 1, and IL-1α, which was not reduced on Day 2, concentrations of cytokine mRNA decreased to Day 3. No bioactivity was detected for GM-CSF, granulocyte colony-stimulating factor or IL-3, but CSF-1, IL-1, IL-6 and TNF-α were detected on Day 1 using bioassays. Changes in concentrations approximately paralleled those for mRNA. The concentrations of mRNA for CSF-1, IL-1, IL-6 and TNF-α were higher on Day 1 of pregnancy than in the uteri of cycling mice 24 h earlier. The data are consistent with previous morphological observations demonstrating the expression of an acute inflammatory response in the mouse uterus after mating. Further, the data demonstrate the expression of genes for CSF-1, GM-CSF, IL-1α, IL-1β, IL-6 and TNF-α is induced in the uterus during mating.

Keywords: mating; interleukin 1; interleukin 6; tumour necrosis factor alpha; colony-stimulating factors; mouse

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M. De, T. R. Sanford and G. W. Wood

Interleukin 1 (IL-1), IL-6 and tumour necrosis factor alpha (TNF-α) are expressed in the mouse uterus on days 1–3 of pregnancy. Cytokine production is temporally associated with the post-mating intrauterine acute inflammatory response. In this study, IL-1, IL-6 and TNF-α were detected in the uterus of pregnant mice from day 3 to day 9, using northern blotting, bioassays and immunocytochemistry. IL-1 bioactivity increased from a low concentration on day 3 to a peak between days 4 and 5 and decreased to low concentrations on days 7 and 8. Blastocyst implantation occurs late on day 4. IL-6 bioactivity was high from day 3 to day 9 and activity was maximal on days 5 and 6. TNF-α bioactivity increased from its lowest concentration on day 3 to a peak on day 8. Although changes in bioactivity concentrations occurred at different times from changes in mRNA concentrations, the changes were approximately parallel. Translation of mRNA into an immunologically detectable product was confirmed using immunocytochemistry with polyclonal anti-cytokine antibodies. We conclude that the cytokines IL-1, IL-6 and TNF-α are produced in the uterus during the periimplantation period of pregnancy in mice. Changes in cytokine concentrations suggested the existence of some form of regulated expression.

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W. F. Rall, M. J. Wood, C. Kirby and D. G. Whittingham

Summary. Eight-cell mouse embryos were cryopreserved by vitrification in a concentrated solution of dimethylsulphoxide, acetamide, propylene glycol and polyethylene glycol. This solution (designated VS1) does not crystallize when cooled to subzero temperatures but instead forms a glassy transparent solid. Embryos were exposed in three steps to a stock VS1 solution or a saline solution containing 90% of the cryoprotectants in the stock VS1 (90% VS1) and then the suspensions were vitrified by rapid cooling in liquid nitrogen. Of 568 embryos vitrified in 90% VS1, 80% developed in vitro and 98 normal fetuses or young (17% of the total) were produced after transfer to pseudopregnant recipients. By contrast, 22% of 153 embryos vitrified in the stock VS1 developed in vitro, but only one normal fetus was obtained after transfer.

These results demonstrate that normal fetuses and young can be produced from embryos cryopreserved by the simple and rapid method of vitrification.

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S. L. Monfort, G. W. Asher, D. E. Wildt, T. C. Wood, M. C. Schiewe, L. R. Williamson, M. Bush and W. F. Rall

This study tested the efficacy of assisted reproduction (synchronization of oestrus and intrauterine artificial insemination (AI)) in contributing to the captive propagation of an endangered species, the Eld's deer (Cervus eldi thamin). Semen was collected from males preselected on the basis of under-represented genotype. Motility of spermatozoa after thawing from ejaculates diluted with BF5F extender (8% glycerol), frozen on dry ice in 0.5 ml straws and stored in liquid nitrogen was 60–70%. Intravaginal progesterone-releasing devices (controlled internal drug release, CIDR-type G) were inserted into 20 adult Eld's deer hinds for 14 days. In all hinds, semen (7.5–10 × 106 motile spermatozoa per uterine horn) was deposited by laparoscopy performed 70 h after removal of the CIDR device. Ovarian activity, before and after AI, was monitored by analysing pregnanediol-3α-glucuronide (PdG) concentrations in voided urine collected three to seven times per week. During the period of CIDR device insertion, urinary PdG profiles were equal to, or above, normal luteal phase concentrations in all hinds. Within 48 h of device withdrawal, PdG concentrations returned to baseline values in 17 of the 20 females, and the onset of behavioural oestrus occurred at this time in 12 hinds. On the basis of sustained increases in urinary PdG, 9 of the 20 hinds were diagnosed as pregnant by 90 days after AI, all of which delivered offspring after a mean gestation of 241.1 days (range, 235–245). Seven singletons (two females, five males) were born alive and survived, and one singleton and one set of twins were stillborn (three females). This is the greatest number of pregnancies and offspring produced in a single AI trial for any endangered mammal. These results demonstrate that genotype preselection can be combined with assisted reproductive technologies, including use of frozen semen, to produce genetically valuable offspring useful for conserving a rare species.