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WV Holt and AR Pickard

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.

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A Medrano, PF Watson and WV Holt

A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.

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JN Garnier, WV Holt and PF Watson

Female wild black rhinoceroses in Zimbabwe were monitored non-invasively using faecal progesterone metabolite analysis and observation of reproductive behaviour. A postpartum period of reproductive inactivity of at least 4 months, followed by a period of 4-7 months of oestrous cyclicity, was detected in six multiparous females. Three-quarters of the oestrous cycles (n = 21) had a total duration (mean +/- SEM) of 26.8 +/- 1 days. Other types of cycle were characterized either by an extended luteal phase, lasting on average twice as long as the normal cycle, or by an extended follicular phase. These extended cycles may have resulted from early embryo loss and heat stress. Female rhinoceroses did not conceive before 8 months after giving birth and some females (n = 2) most likely aborted after 3.0-3.5 months of gestation. The detected period of cyclic oestrus occurred between May and March in females (n = 9), and there was a 3 month extended interoestrous interval in nulliparous females during the period of decreasing daylengths that can be presumed to be the period of poorest fertility for the black rhinoceros under tropical latitudes. In contrast, the period of optimum fertility in the Southern hemisphere coincided with the late spring and early summer, and corresponded to the early rainy season. As a result, a higher incidence of births was detected in the late rainy season, providing the lactating female with the most suitable environment in terms of nutritional requirements.

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AR Pickard, T Abaigar, DI Green, WV Holt and M Cano

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.

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CE Green, J Bredl, WV Holt, PF Watson and A Fazeli

After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.

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T Abaigar, M Cano, AR Pickard and WV Holt

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.

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A Fazeli, RM Elliott, AE Duncan, A Moore, PF Watson and WV Holt

Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.