Upgrading mouse health and welfare: direct benefits of a large-scale rederivation programme.

We report the outcome of a 30-month programme to rederive 310 specific pathogen-free mouse strains to populate a new individually ventilated cage barrier facility at the Mary Lyon Centre (MLC), Medical Research Council (MRC) Harwell. The mice were rederived in a self-contained quarantine suite and embryo-recipient females were health-screened to assess microbiological status, before moving their offspring into the new facility. The MLC currently houses approximately 49,000 mice in about 9750 cages and we have 30 months of follow-up health screen data. Embryo rederivation and hysterectomy have high safety margins; however, the precaution of performing the programme in isolators facilitated the containment and decontamination of two mouse hepatitis virus (MHV) infection outbreaks. Rederivation of the colony has eliminated endemic MHV, mouse adenovirus type 2 (MAV-2), Theiler's murine encephalomyelitis virus, pinworms, intestinal protozoa, Pasteurella pneumotropica, Helicobacter spp. and mites. The improvements in microbiological status have had notable benefits for mouse health and welfare and the science at MRC Harwell. Previously important clinical entities such as sudden death associated with lactation ileus in C3H/HeH mice, early weight loss associated with inflammatory bowel disease in B6-TgN(HDexon1)61Gpb and B6TgN-(HD82Gln)81Dbo (Huntington) mice and early weight loss in male mice mutagenized with N-ethyl-N-nitrosourea have been markedly reduced or eliminated.

Patterns of fetal growth within Large White x Landrace and Chinese Meishan gilt litters at three stages of gestation.

Low birthweight piglets have an increased incidence of mortality and morbidity. As there are few opportunities to remedy the detrimental consequences of low birthweight after birth, it is important to understand the nature of fetal growth retardation and to identify when low birthweight fetuses deviate from the growth trajectory of their normally grown siblings. The aims of this study were to identify the nature, timing and possible causal factors influencing inadequate fetal growth in Large White x Landrace (LW) and Chinese Meishan (MS) gilts at three stages of pregnancy. Thirty-six per cent of litters contained inadequately grown fetuses. Both intrauterine-growth-restricted (IUGR) and small-for-gestational-age (SGA) fetuses could be identified as early as Day 30 in MS and LW litters and the percentage of litters containing inadequately grown fetuses was similar throughout gestation. MS fetuses, placentas and piglets had less within-litter variation in weight at all stages studied. Inverse relationships were observed between litter size and both minimum and mean weights of MS neonates. No other relationships between fetal size and either uterine position or litter size were observed.

Hormonal characterization of the reproductive cycle and pregnancy in the female Mohor gazelle (Gazella dama mhorr).

The oestrous cycles of seven captive Mohor Gazelles (Gazella dama mhorr) were investigated. Hormone profiles obtained from faecal samples collected each day from cyclic females indicated that the mean duration of the oestrous cycle was 18.62 +/- 0.26 days (range 16-22 days; n = 37 oestrous cycles). No inter-individual differences in the concentration of faecal progestagen metabolites excreted were observed, but mean faecal oestrogen excretion during both the luteal and inter-luteal phases of the oestrous cycle varied among females (P < 0.001 and P = 0.070, respectively). Oestrous cycles were synchronized using controlled internal drug release (CIDR) devices, before natural mating with an intact male. Concentrations of faecal progestagen metabolites remained approximately constant for the first 10 weeks of gestation (mean +/- SEM = 4048 +/- 407 ng g(-1) faeces), before increasing to a mean of 23 556 +/- 1176 ng g(-1) faeces. Two of seven female gazelles conceived immediately after removal of the CIDR device, a similar proportion to that conceived at the postpartum oestrus under natural conditions. Life history data for these individuals indicated that the mean time to conception in female gazelles is positively correlated with peak values in the ratio of excreted oestrogen : progestagen during the inter-luteal period of their oestrous cycles (R(2) = 0.58; P < 0.05). This finding indicates that interactions between steroid production and metabolism may influence the likelihood of conception occurring in this species.

Use of computer-assisted sperm motility assessment and multivariate pattern analysis to characterize ejaculate quality in Mohor gazelles (Gazella dama mhorr): effects of body weight, electroejaculation technique and short-term semen storage.

Subjective and objective semen assessments were performed on 18 male Mohor gazelles (Gazella dama mhorr). Sperm motility assessments combined with sperm plasma membrane and acrosomal integrity evaluations were undertaken as part of a captive breeding programme. The primary objective was to test methodology for short-term preservation of gazelle semen for artificial insemination (storage in N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulphonic acid-Tris diluent (TEST) for up to 96 h at 17 degrees C). However, the secondary objective was to investigate phenotypic and genotypic influences on semen quality within this small population, which was established in 1971 with only 12 genetic founders. Sperm motility was measured by computer-assisted semen assessment and the data were analysed using a pattern analysis technique to detect and quantify naturally occurring sperm subpopulations within the semen samples. Four sperm subpopulations distinguishable by their motion characteristics were detected. The relative frequencies of two subpopulations (population 2: highly motile, non-linear; and population 4: poorly motile, non-linear) in fresh semen were correlated with the maximum voltage used during electroejaculation. The frequency of subpopulation 2 was negatively correlated with maximum voltage (r = -0.875, P < 0.0001) and the frequency of subpopulation 4 was positively correlated (r = 0.727, P < 0.005). The frequencies of all subpopulations varied significantly among the animals sampled (chi-squared = 2577.6, degrees of freedom = 54, P < 0.0001) and subpopulation 4 was also correlated with body weight (r = -0.59, P < 0.005). Semen stored at 17 degrees C retained motility, plasma membrane and acrosomal integrity for 48 h, but these measures decreased thereafter. The frequency of a sperm subpopulation showing uncoordinated but active motility increased significantly over the first 48 h and then decreased.

Non-invasive monitoring of reproductive status in wild mongoose lemurs (Eulemur mongoz).

Endocrine profiles were investigated in wild and captive mongoose lemurs (Eulemur mongoz; Lemuridae) by analysing faecal progestagens and oestrogens. Oestrous cycle characterization was not possible, as most females appeared to conceive during the first oestrus of the breeding season. Conception was preceded by a pseudo-oestrus with no discernible luteal phase. Pseudo-oestrus and oestrus identification was possible by investigating the oestrogen:progestagen ratio. Pregnancy was reliably determined approximately 47 days after conception, when progestagen and oestrogen excretion increased above breeding season concentrations. Gestation was further characterized by high progestagen concentrations and a decline in oestrogen excretion 70-80 days after conception. Post partum, progestagens declined, but oestrogen excretion increased to exceed breeding season concentrations. In the wild group, a nulliparous daughter conceived while still a member of her natal group and aborted after 70-80 days of pregnancy at which time progestagens had declined but oestrogens remained high. Comparisons with other strepsirhine primates suggest that pseudo-oestrus followed by conception at first oestrus may be prevalent in lemurs. Gestational progesterone profiles vary between species, but a delayed increase in oestrogens during pregnancy could be common to all strepsirhines, although oestrogen levels during the final trimester of pregnancy differ between lemurs and lorises. Methodological investigations showed that prolonged storage of faeces in ethanol is viable and that the presence of undigested vegetable matter in the faecal pellets had no effect on the interpretation of hormone profiles.

Role of reproductive technologies and genetic resource banks in animal conservation.

In combination with modem reproductive technologies, there is potential to use frozen and stored germplasm (genetic resource banks) to support conservation measures for the maintenance of genetic diversity in threatened species. However, turning this idea into reality is a complex process, requiring interdisciplinary collaboration and clearly defined goals. As the number of species deserving the attention of conservation scientists is overwhelmingly large, yet detailed knowledge of reproductive physiology is restricted to relatively few of them, choosing which species to conserve is one of the most difficult issues to be tackled. Besides the direct application of technologically advanced reproductive procedures, modern approaches to non-invasive endocrine monitoring play an important role in optimizing the success of natural breeding programmes. Through the analysis of urine and faecal samples, this type of technology provides invaluable management information about the reproductive status of diverse species. For example, it is possible to diagnose pregnancy and monitor oestrous cycles in elephants and rhinos without causing stress through restraint for sample collection. In this review, we identify the potential contribution of reproductive biology and genetic resource banks to animal conservation, but also highlight the complexity of issues determining the extent to which this potential can be achieved.

Non-invasive diagnosis of pregnancy in wild black rhinoceros (Diceros bicornis minor) by faecal steroid analysis.

The objective of this study was to determine whether faecal progestagen measurement could be used to diagnose pregnancy in wild black rhinoceros cows. Immunoreactive 20alpha-progestagens were measured in faecal samples collected regularly (one or two times times per week) from pregnant and non-pregnant wild black rhinoceros females (n = 6) in Zimbabwe. Fresh dung piles deposited by the study animals were serially sampled during prolonged periods of tracking with local game scouts. Samples were stored frozen, and dried prior to methanol extraction. Immunoreactivity in faecal extracts was measured with a 20alpha-dihydroprogesterone enzyme immunoassay and was shown to reflect circulating progesterone concentrations. Mean concentrations of faecal 20alpha-progestagens during each month of gestation were significantly higher than faecal concentrations in non-pregnant animals (P<0.05), except during the second month of gestation. Faecal 20alpha-progestagens remained 5-10 times higher than concentrations in non-pregnant animals from the 4th to 15th month of gestation. It was concluded that regular non-invasive reproductive monitoring of black rhinoceros in the wild was possible and that pregnancy could be accurately diagnosed from the measurement of 20alpha-progestagens in faecal samples. The use of this technique in wild black rhinoceros populations will offer new perspectives for in situ management of this endangered species.

Expression of tissue inhibitor of metalloproteinase-1 protein and messenger ribonucleic acid by the oviduct of cyclic, early-pregnant, and ovariectomized steroid-treated gilts.

It has been suggested that tissue inhibitor of metalloproteinases (TIMP)-1 has a role in reproductive tissues, regulating tissue remodeling or enhancing embryonic development. Oviductal TIMP-1 mRNA levels and protein expression were examined in gilts during the estrous cycle and early pregnancy and in steroid-treated ovariectomized (OVX) gilts by explant culture, two-dimensional SDS-PAGE and fluorography, dot-blot hybridization, immunoblot analysis, RIA, and immunocytochemical studies. TIMP-1 mRNA levels in the oviduct during the estrous cycle were greater (p < 0.02) on Days 2, 15, and 18 than on other days examined, and analysis of oviductal functional segments indicated an effect of day (p < 0.003), an effect of segment (p < 0.007), and a day x segment effect (p < 0.03). The level of TIMP-1 mRNA was greater (p < 0.003) in the isthmus (I) on Day 2 than in the ampulla (A) or infundibulum (INF) or on other days examined (0 and 12). In steroid-treated OVX gilts, an effect of treatment with estradiol valerate (EV) + progesterone (P4) was shown with increased (p < 0.003) TIMP-1 mRNA levels. De novo synthesis of TIMP-1 protein was found throughout the estrous cycle and early pregnancy in all functional segments, but protein expression was greater in the I and greatest on Day 2. In steroid-treated OVX gilts, TIMP-1 protein synthesis was greatest in the I regardless of treatment, but with increased intensity after EV+P4 treatment. TIMP-1 protein was found in oviductal flushings during the estrous cycle and early pregnancy, and in steroid-treated OVX gilts regardless of day, status, or treatment. Differences in TIMP-1 concentrations in oviductal fluid were found by day (p < 0.001), with breed differences detected between the Meishan and standard Western breeds. TIMP-1 protein was immunolocalized primarily to luminal epithelium of the INF, A, and I on all days of the estrous cycle and early pregnancy and to some cells in the stroma and blood vessel walls. Staining intensity correlated with TIMP-1 protein levels in oviductal flushings. The role of TIMP-1 in the oviduct remains to be established.

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.

Comparative studies of conceptus-endometrial interactions in Large White x Landrace and Meishan gilts.

Uterine and conceptus function were compared in mated Meishan (MS) gilts and Large White x Landrace (LW x L) gilts (n = 18 breed-1) on Days 11-15, inclusive, after oestrus. Comparisons of individual blastocysts recovered on Day 11 and Day 12 revealed a higher overall embryo survival in MS gilts than in LW x L gilts (100.7 +/- 5.0% v. 69.5 +/- 12.1%; P < 0.05). Embryo survival was higher on Day 11 than on Day 12 (98.6 +/- 6.0% v. 71.6 +/- 12.6%; P < 0.05), with most of the embryo loss between these days occurring in LW x L gilts. MS conceptuses secreted less oestradiol-17 beta and radiolabelled protein on both days, but these differences were not significant. The within-litter variability in the secretion of oestradiol-17 beta and radiolabelled protein by individual conceptuses did not differ significantly between the breeds. Concentrations of epidermal growth factor in uterine rinsings were lower in MS gilts than in LW x L gilts on all days studied. However, two-dimensional polyacrylamide gel electrophoresis and fluorography did not provide evidence of additional breed differences in the profile of endometrial secretory proteins. Consistent temporal changes in the profile of the conceptus secretory proteins were observed among all LW x L gilts and among most of the MS gilts. However, conceptuses cultured from 2 of 5 Day-12 MS gilts secreted a major basic protein which was not evident in other conceptus cultures until Day 14. Similarly, antiviral activity was detected in some cultures of Day-13 MS conceptuses, but was absent from all Day-13 LW x L conceptus cultures. The results also revealed a positive relationship between ovulation rate and the incorporation of radiolabel into endometrial secretory proteins, which was independent of breed.

A comparison of recruitment methods for an osteoarthritis exercise study.

OBJECTIVE: Patient recruitment often involves a great deal of effort and cost. We sought to determine successful recruitment methods for an osteoarthritis (OA) exercise study, and to subsequently focus on using those approaches. METHODS: Eleven methods were developed to recruit subjects for this large research project. Financial constraints ensured that we focused on low-cost measures. Over a 20-month period, the numbers of both total respondents and eventual participants were recorded. RESULTS: Responses were recorded for 263 individuals, and 108 subjects entered the study. The most successful recruitment method was via physician referrals from affiliated clinics. In general, methods that were free of direct cost seemed no less successful than those requiring expenditures. CONCLUSION: Successful subject recruitment, even for a large study, may not require costly advertising. Direct contact and frequent reminders rendered to our own clinics proved most effective, but other free and low-cost approaches were also of benefit.

Differences in follicular aromatase activity between Meishan and large-white hybrid gilts.

Two experiments were carried out in which follicular aromatase activity was compared between Meishan and Large-White hybrid gilts. In Expt 1, preovulatory follicles (n = 10 largest per animal) were recovered from Meishan and Large-White hybrid gilts (n = 5 per breed) on the day before predicted onset of behavioural oestrus, and the granulosa cells and theca tissue incubated to determine aromatase activity. Follicles recovered from Meishan pigs were smaller (P < 0.01) and contained fewer granulosa cells (P < 0.05), but follicular oestradiol content of the breeds was similar (P > 0.1). Aromatase activity was higher in the theca tissue (P < 0.05) and tended to be higher in the granulosa cells recovered from Meishan follicles (P = 0.065). In Expt 2, granulosa cell aromatase activity was investigated during the early follicular phase (estimated day 16 of cycle) in Meishan and Large-White hybrid gilts (n = 6 and 5, respectively). The number of follicles > or = 1 mm diameter recovered per animal was 171 for both breeds (P > 0.1), whereas the number of follicles > or = 2 mm diameter was 65 and 101 (P < 0.05) from Meishan and Large-White hybrid gilts, respectively. The mean diameter of all follicles recovered was smaller in the Meishan gilts (P < 0.001). Overall, neither the number of granulosa cells per follicle, as indicated by DNA estimation, nor the oestradiol content differed between the breeds at this time (P > 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Presentation of Class I antigens on T cells can induce the generation of antigen-specific CTL.

Cytotoxic T lymphocyte (CTL) lines generally require a source of specific antigen to continue to proliferate in vitro. We previously showed that populations of mononuclear cells grown in medium containing interleukin 2 (IL 2) and composed largely of activated T cells were able to present Class I alloantigen to CTL lines. On the basis of these findings we were interested to know whether T cells themselves were able to present antigen or whether this was a function of the small number of contaminating non-T cells. To answer this question, populations of activated or resting mononuclear cells were rigorously depleted of non-T cells before use as antigen-presenting cells. We observed that populations composed of greater than 99% T cells were able to support the differentiation of antigen-specific CTL. These results were confirmed by using cells from an established T cell line. The proliferation of the same lines, however, was less than that of lines grown in the presence of antigen-presenting cells containing some non-T cells. These results suggest that although T cells can present Class I alloantigens to CTL, they may be less effective in triggering cell division than populations containing a source of non-T cells.

Expression of phosphorylcholine-specific B cells during murine development.

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.

Assessing B cell diversification by antigen receptor and precursor cell analysis.

A major element in the understanding of B cell specificity diversification is the extent of diversity present in mature and in developing B cell populations. Two general methods are currently used for assessing the specificity repertoire: (a) the enumeration of cells whose receptors can bind a specific antigen, and (b) the enumeration of cells which can respond to antigenic stimulation by antibody-forming cell clone production. Our laboratory has utilized the latter method to establish the frequency of B cells responsive to a wide variety of antigenic determinants. The findings indicate that: (a) the primary murine B cell specificity repertoire probably includes more than 10(7) clonotypes; (b) some clonotypes are represented by numerous B cells (40,000 TEPC 15 precursors per BALB/c mouse) while most are represented by fewer than to B cells per mouse; (c) the acquisition of the repertoire is apparently antigen-independent since germfree mice have repertoires similar to conventional mice and secondary B cells are easily distinguished from primary B cells; (d) the neonatal repertoire appears to contain only 10(4) clonotypes at birth, each represented by perhaps 200-400 cells; (e) the diversification process from neonatal to adult repertorie appears highly ordered and reproducible. Antigen binding cell studies have now used in conjunction with the splenic focus assay in an attempt to correlate these two techniques. The results indicate that the efficiency of the splenic focus assay used for precursor cell anlysis it 4-5% for both primary and secondary B cells and is similar to the percent of donor B cells lodged in recipient spleens. For certain antigens (DNP-BSA) the number of antigen-binding cells can represent 4% of the total B cells, and this number directly correlates with the concentration of antigen used; stimulation, on the other hand, appears to have an affinity threshold achieved by only 0,02% of the DNP-specific B cells. In contrast, PC-BSA antigen-binding cell and splenic focus precursor cell frequencies are identical. These findings are interpreted to indicate that antigen-binding cell analyses confirm the validity of the calculations used to estimate precursor frequencies in the splenic focus technique. However, for some antigens binding to cell receptors, one detects a large number of cells belonging either to a nonstimulatable B cell subclass or whose receptor affinity is too low to permit stimulation.