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Y. Gao and R. V. Short

Paraffin blocks containing either no steroid, 150 mg RU486 kg−1, 500 mg methyl testosterone kg−1 or 1500 mg methyl testosterone kg−1 were fed to wild mice (Mus musculus in addition to the standard laboratory diet in four large (3 m × 3 m) outdoor pens for six months over the summer. The RU486 bait was provided for only 3 days every 18 or 21 days, whereas the methyl testosterone bait was available continuously. From a foundation stock of 20 mice (nine male, eleven female) in each pen, the population had increased to 253 (control), 72 (RU486), 249 (low methyl testosterone concentration) and 103 (high methyl testosterone concentration) at the end of six months, when 17%, 4%, 32% and 13% of the mature females were pregnant in the respective treatment groups. There was little evidence of an increase in the incidence of injuries in the androgen-treated animals. Daily estimation of water consumption in the pens proved to be a good non-invasive way of monitoring population growth during the course of the experiment. Intermittent feeding with a low concentration of RU486 appeared to be much more successful in inhibiting reproduction than continuous feeding with a high concentration of methyl testosterone, and it therefore offers a new method for controlling feral mouse populations.

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Y. Gao and R. V. Short

Three synthetic steroids were evaluated as potential chemosterilants for rodent control. Ethinyl oestradiol, methyl testosterone or Org 5933, a synthetic gestagen, were incorporated into paraffin blocks containing cereal grains and offered to laboratory rats and mice in addition to their standard laboratory diet. Ethinyl oestradiol (50 mg kg−1 paraffin block) was highly unpalatable to female rats, and the amount of steroid ingested was not sufficient to interfere with their oestrous cycles or inhibit ovulation. Methyl testosterone (5000 mg kg−1 paraffin block), although not as palatable as untreated blocks, was effective in inducing almost immediate infertility in female rats and mice at an ingested dose of about 35 μg g−1 body weight day−1. This infertility persisted throughout the duration of treatment, and lasted for several weeks after the cessation of treatment. Male rats became infertile after 3 months of treatment owing to suppression of spermatogenesis. Female rats developed a specific aversion to methyl testosterone when they were pregnant or lactating; it was therefore not possible to masculinize the brains of their female offspring. In mice, the androgen treatment induced high levels of aggression in the females so that they fought with males and with one another. One female died of her wounds. Org 5933 (4 mg kg−1 paraffin block) was highly palatable to female rats and mice, and at doses of about 420 ng g−1 body weight day−1 was effective in inhibiting ovulation in rats within 3 to 4 days after the start of treatment. This infertility persisted throughout the duration of treatment, and the animals conceived within 5 days of cessation of treatment. A dose of about 930 ng g−1 body weight day−1 was not completely effective in inhibiting ovulation in mice, but females that became pregnant during treatment gave birth to dead young. When the gestagen was given to female rats and mice in the last few days of pregnancy, the duration of gestation was significantly prolonged, and most young were born dead; some of the females also died in labour. The gestagen did not appear to inhibit lactogenesis, since the few animals that gave birth to live young reared them normally for the first 5 days of life. These results show that either methyl testosterone or Org 5933 in paraffin blocks could perhaps be used as a chemosterilant for the control of rat and mouse populations. The optimal strategy would be to use the chemosterilant when the population density of rodents was lowest, for example at the end of the winter, or following a poisoning campaign with conventional rodenticides, thereby preventing the survivors from reproducing and spreading genetic resistance to the poison.

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Y. Gao and R. V. Short

The intermittent use of an antigestagen could prove to be a very effective way of controlling the fertility of rats and mice in the wild. This concept was tested by giving paraffin wax blocks containing cereal grains and the antigestagen Mifepristone, RU486 (150 mg kg−1 block) to male and female laboratory rats and mice in a series of free-choice feeding experiments. There was no significant difference in the consumption of blocks with or without RU486, showing that it was completely palatable to rats and mice, and no aversion developed following refeeding. The average consumption of RU486 by rats was 11 mg kg−1 day−1; mice consumed 37 mg kg−1 day−1. All the females showed persistent oestrous vaginal smears throughout the treatment. When male and female rats and mice were given continuous access to treated paraffin blocks for 30 days, no conceptions occurred. At the end of this time, there was a significant increase in ovarian weight in the treated rats and mice, but no difference in testicular weight. Treated blocks were given to rats for 3 days every 21 days for a total of 115 days. Four dead litters were produced following the first antigestagen treatment on day 21, but no more litters were produced and no treated rats were pregnant when autopsied on day 115. Mice were initially treated for 3 days every 21 days, but some animals continued to produce live young on this schedule. The treatment period was therefore reduced to 3 days every 18 days and no more litters were produced and none of the treated females was pregnant at autopsy. The antigestagen RU486 shows considerable promise as a chemosterilant for the control of fertility in female rats and mice. Intermittent administration every 18 days (mice) or 21 days (rats) in free-choice feeding trials completely inhibited reproduction.

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Y. S. Xu, H. Y. Wang, G. Q. Zeng, G. T. Jiang and Y. H. Gao

Summary. During the follicular phase of bactrian camels, basal concentrations of LH were 2·7 ± 1·2 ng/ml. By 4 h after insemination peak values of 6·9 ± 1·0 ng/ml occurred. In addition, a smaller LH peak (5·4 ± 2·5 ng/ml) appeared 1 day before regression of the follicle began in unmated camels. During the follicular phase peripheral plasma progesterone values were low (0·36 ± 0·28 ng/ml), but values increased to reach 1·73 ± 0·74 ng/ml at 3 days and 2·4 ± 0·86 ng/ml, at 7 days after ovulation. Plasma oestradiol-17β concentrations were 26·8 ± 9·0 pg/ml during the follicular phase and 30·8 ± 5·1 pg/ml when the follicle was maximum size. Values fell after ovulation but rose to 29·8 ± 6·5 pg/ml 3 days later.

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D. Y. Gao, J. J. McGrath, Jun Tao, C. T. Benson, E. S. Critser and J. K. Critser

A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8–10 μm diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (L p) found to be 1.05, 0.45 and 0.26 μm min−1 atm−1 at 30°C, 22°C and 12°C, respectively. The corresponding activation energy, E a, for L p was calculated to be 13.0 kcal mol−1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium. The second advantage is that the time required for mixing the original cell suspension and perfusion medium is minimized, allowing for accurate control of the extracellular osmolality and ensuring accuracy of the subsequent mathematical formation. This technique also has wide applicability in determining the membrane transport properties of mammalian oocytes, embryos and other cell types.

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C L Lu, J Yan, X Zhi, X Xia, T R Wang, L Y Yan, Y Yu, T Ding, J M Gao, R Li and J Qiao

Fertility preservation is an important type of frontier scientific research in the field of reproductive health. The culture of ovarian cortices to i) initiate primordial follicle growth and ii) procure developing follicles for later oocyte maturation is a promising fertility preservation strategy, especially for older women or cancer patients. At present, this goal remains largely unsubstantiated in primates because of the difficulty in attaining relatively large follicles via ovarian cortex culture. To overcome this hurdle, we cultured macaque monkey ovarian cortices with FSH, kit ligand (KL), basic fibroblast growth factor (bFGF), and/or epidermal growth factor (EGF). The various factors and factor combinations promoted primordial follicle development to different extents. Notably, both bFF (bFGF, 100 ng/ml and FSH, 50 ng/ml) and KF (KL, 100 ng/ml and FSH, 50 ng/ml) contributed to the activation of primordial follicles at day 12 (D12) of culture, whereas at D18, the proportions of developing follicles were significantly higher in the bFF and KF groups relative to the other treatment groups, particularly in the bFF group. Estradiol and progesterone production were also highest in the bFF group, and primary follicle diameters were the largest. Up until D24, the bFF group still exhibited the highest proportion of developing follicles. In conclusion, the bFGF–FSH combination promotes nonhuman primate primordial follicle development in vitro, with the optimal experimental window within 18 days. These results provide evidence for the future success of human ovarian cortex culture and the eventual acquisition of mature human follicles or oocytes for fertility restoration.