A hormone priming regimen and hibernation affect oviposition in the boreal toad (Anaxyrus boreas boreas).

Declines of the southern Rocky Mountain population of boreal toad (Anaxyrus boreas boreas) have led to the establishment of a captive assurance population and reintroduction program, in an attempt to preserve and propagate this geographically isolated population. One of the unique adaptations of this species is its ability to survive in cold environments by undergoing long periods of hibernation. In captivity, hibernation can be avoided altogether, decreasing morbidity caused by compromised immune systems. However, it is not entirely clear how essential hibernation is to reproductive success. In this study, the effects of hibernation versus nonhibernation, and exogenous hormones on oviposition, were examined in boreal toad females in the absence of males. In the summers of 2011 and 2012, 20 females housed at Mississippi State University were treated with a double priming dose of hCG and various ovulatory doses of hCG and LH-releasing hormone analog but denied hibernation. Exogenous hormones, in the absence of hibernation, could not induce oviposition over two breeding seasons (2011-2012). In contrast, during the summer of 2012 and 2013, 17 of 22 females (77%) housed at the Native Aquatic Species Restoration Facility (Alamosa, CO, USA) oviposited after they were treated with two priming doses of hCG (3.7 IU/g each) and a single ovulation dose of hCG (13.5 IU/g) and LH-releasing hormone analog (0.4 µg/g) after hibernation. There was a significant difference in oviposition between females that were hibernated and received hormones (2012, P < 0.05 and 2013, P < 0.01) compared to hibernated control females. In 2013, 12 of 16 remaining Mississippi State University females from the same group used in 2011 and 2012 were hibernated for 1, 3, and 6 months, respectively and then treated with the same hormone regimen administered to females at the Native Aquatic Species Restoration Facility. Together, hibernation and hormone treatments significantly increased oviposition (P < 0.05), with 33% of females ovipositing. These results suggest that (1) hibernation is a key factor influencing oviposition that cannot be exclusively circumvented by exogenous hormones; (2) females do not require the presence of a male to oviposit after hormone treatments; and (3) longer hibernation periods are not beneficial for oviposition. The hormonal induction of oviposition in the absence of males and shorter hibernation periods could have important captive management implications for the boreal toad. Furthermore, the production of viable offspring by IVF where natural mating is limited could become an important tool for genetic management of this boreal toad captive population.

Nutrient and mineral composition during shoot growth in seven species of Phyllostachys and Pseudosasa bamboo consumed by giant panda.

During the annual period of bamboo shoot growth in spring, free-ranging giant pandas feed almost exclusively on the shoots while ignoring the leaves and full- height culm. Little is known about the nutritional changes that occur during bamboo shoot growth, if nutritional changes differ among species, or how these changes might influence forage selection. Our objective was to examine the nutrient and mineral composition during three phases of shoot growth (<60, 90-150 and >180 cm) for seven species of bamboo (Phyllostachys (P.) aurea, P. aureosulcata, P. bissetii, P. glauca, P. nuda, P. rubromarginata, Pseudosasa japonica) fed to captive giant pandas at the Memphis Zoo. Total dietary fiber content of bamboo shoots increased (p < 0.0001) from an overall species average of 61% dry matter (DM) at < 60 cm to 75% DM at shoot heights > 180 cm, while crude protein, fat and ash exhibited significant declines (p < 0.05). Phyllostachys nuda had the overall greatest (p = 0.007) crude protein (21% DM) and fat (4% DM) content, and lowest overall total fibre (61% DM) content compared to the other species examined. In contrast, Pseudosasa japonica had the overall lowest crude protein and fat, and relatively higher fibre content (9%, 3% and 74% respectively). Concentrations of Zn and Fe were highest in shoots <60 cm (10-50 µg/g DM) and decreased (p < 0.05) during growth in all species examined. Concentrations of Ca, Cu, Mn, Na and K varied among species and were largely unaffected by growth stage. Due to their higher concentrations of nutrients and lower fibre content in comparison to culm and leaf, bamboo shoots should be a major component of captive giant panda diets when available.

Artificial fertilization for amphibian conservation: current knowledge and future considerations.

Amphibian populations in the wild are experiencing massive die-offs that have led to the extinction of an estimated 168 species in the last several decades. To address these declines, zoological institutions are playing an important role in establishing captive assurance colonies to protect species in imminent danger of extinction. Many of the threatened species recently placed into captivity are failing to reproduce before they expire, and maintaining founder populations is becoming a formidable challenge. Assisted reproductive technologies, such as hormone synchronization, gamete storage and artificial fertilization, are valuable tools for addressing reproductive failure of amphibians in captive facilities. Artificial fertilization has been commonly employed for over 60 years in several keystone laboratory species for basic studies in developmental biology and embryology. However, there are few instances of applied studies for the conservation of threatened or endangered amphibian species. In this review, we summarize valuable technological achievements in amphibian artificial fertilization, identify specific processes that need to be considered when developing artificial fertilization techniques for species conservation, and address future concerns that should be priorities for the next decade.

Species-specific sperm-egg interaction affects the utility of a heterologous bovine in vitro fertilization system for evaluating antelope sperm.

The purpose of this study was to evaluate cryopreserved fringe-eared (FE) oryx (Oryx gazella callotis) sperm function using a heterologous in vitro fertilization (IVF) system previously developed to study scimitar-horned (SH) oryx (Oryx dammah) spermatozoa. Semen was collected by electroejaculation from FE oryx (n = 2) and SH oryx (n = 2), evaluated immediately postcollection, and cryopreserved. Thawed spermatozoa were evaluated for motility, forward progression, and acrosomal status immediately post-thaw, after Percoll-separation, and 1, 2, 3, and 8 h after culture in IVF medium. In vitro-matured cow oocytes (n = 924) were inseminated with either domestic bull, FE, or SH oryx spermatozoa and after an 8-h coincubation period, half the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were placed into in vitro culture and evaluated for cleavage after 48 h. Overall, there were no between-species differences in sperm motility and acrosome integrity. However, an effect of time (P < 0.05) and a species-by-time interaction (P < 0.05) were detected for both parameters. Penetration, male pronuclear formation, and embryo cleavage were high (>90%, >85%, and >70%, respectively) for oocytes inseminated with domestic bull and SH oryx spermatozoa and did not differ (P > 0.05) between species. In contrast, very few oocytes (2.8%, 4 of 141) inseminated with FE oryx sperm were penetrated. Cleavage was rare (8.0%, 16 of 200) in oocytes inseminated with FE oryx spermatozoa and did not differ (P > 0.05) from that in parthenogenetic controls (4.2%, 3 of 72). Furthermore, FE oryx spermatozoa were incapable of penetrating zona-free cow oocytes. These results indicate that species-specific differences in gamete interaction may exist even between very closely related nondomestic bovids.

Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro.

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0-100 microgram/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0-100 microgram/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0-100 microgram/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0-50 microgram/ml had no effect on sperm penetration rates; however, compared with the control, 100 microgram/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10-100 microgram/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24-29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19-34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig.

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.

Secreted proteins of the oviduct.

During late follicular development and estrus, the mammalian oviduct undergoes specific physiological and biochemical modifications which contribute to an optimization of the microenvironment for fertilization and early cleavage-stage embryonic development. These changes appear to be hormonally regulated by ovarian steroids, most importantly, estrogen. The hundreds of macromolecules found within the oviductal lumen are contributed by selective serum transudation and active biosynthesis and secretion from nonciliated epithelial cells. Recent studies have indicated temporal and regional (infundibulum, ampulla and isthmus) differences in steady-state levels of specific mRNAs and in de novo protein synthesis and secretion by the oviduct. One protein synthesized de novo, the estrogen-dependent oviductal secretory glycoprotein (OSP), has been shown to be unique to the oviduct and is conserved across a number of mammalian species. This protein associates with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos. OSPs have been shown to enhance sperm binding and penetration in oocytes and may regulate development in early preimplantation embryos. Other regulatory molecules, protease inhibitors, growth factors, cytokines, binding proteins, enzymes and immunoglobulins have been identified in the oviductal microenvironment. The identification and potential roles for oviduct-secreted proteins will be reviewed and discussed. Current research focuses on continued identification and characterization of specific oviductal proteins and a determination of the molecular basis of their interactions with the oocyte, sperm or embryo.

Identification and localization of plasminogen activator inhibitor-1 within the porcine oviduct.

The porcine oviduct synthesizes de novo and secretes a number of proteins into culture medium, many of which are unidentified. The objectives of the present study were to 1) semipurify and identify a M(r) 45 000 secreted protein of the oviduct, 2) examine its synthesis within the three functional segments (infundibulum, ampulla, and isthmus), and 3) evaluate its distribution throughout the oviduct. Oviductal tissue was collected during early pregnancy, divided into functional segments, and subsequently cultured. Medium was collected, and the M(r) 45 000 protein was concentrated by gel-filtration chromatography. The semipurified protein was transferred onto a polyvinylidene fluoride membrane and subjected to N-terminal amino acid analysis. The 26-amino acid sequence was 96% identical to that of pig plasminogen activator inhibitor (PAI)-1. Analysis by 1-dimensional SDS-PAGE and fluorography of rabbit anti-human PAI-1-immunoprecipitated product confirmed PAI-1. Subsequent 2-dimensional SDS-PAGE and fluorographic analyses of media revealed greater PAI-1 synthesis by the isthmus than by the ampulla or infundibulum. PAI-1 was immunolocalized throughout the oviduct and was heavily concentrated in the apical region of epithelial cells. Immunogold electron microscopy localized PAI-1 within putative secretory granules in the epithelial apical region and also associated with cilia in the isthmus. Isthmic PAI expression suggests a crucial role in protecting the preimplantation embryo from proteolytic degradation as well as in regulation of extracellular matrix turnover and remodeling.

Identification of insulin-like growth factor I in bovine seminal plasma and its receptor on spermatozoa: influence on sperm motility.

Insulin-like growth factor I (IGF-I) has been identified in human seminal plasma. This study was conducted to determine whether IGF-I is present in bovine seminal plasma, whether sperm cells express the IGF-I receptor (IGF-IR), and whether IGF-I affects sperm motility. Semen samples were collected from bulls by electroejaculation and maintained at 37 degrees C, and motility of sperm was assessed. After centrifugation to separate sperm cells from seminal plasma, the seminal plasma was submitted to a validated heterologous RIA for IGF-I. Significant concentrations of IGF-I (116.29 +/- 40.83 ng/ml expressed as mean +/- SD) were measured in bovine seminal plasma. Sperm cells were washed with buffer and subjected to either radioreceptor assay (RRA) or immunocytochemistry (IC). RRA revealed a single high affinity for the IGF-IR with a Kd of 0.83 nM as determined by the computer program LIGAND. IC, using three monoclonal antibodies, localized the IGF-IR to the acrosomal region of the sperm. Computer-assisted sperm-motion analysis was used to determine the effects of IGF-I and IGF-II on bovine sperm motility parameters. Both IGF-I and IGF-II increased sperm motility and straight-line velocity (p < 0.05) relative to the control. The presence of IGF-IR on sperm, the presence of IGF-I in semen, and the ability of IGF-I to stimulate sperm motility provide evidence that the IGF system may be involved in the fertilization process in the bovine 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.

Oviductal regulation of fertilization and early embryonic development.

During the period of late follicular development and the first four days of the oestrous cycle, the oviduct occupies a central role in the establishment of pregnancy. Oviductal function is regarded as being either 'passive' or biologically active, providing an environment that sustains and enhances fertilization and early cleavage-stage embryonic development. Recent reports have focused on this microenvironment and shown that ovarian steroids induce marked morphological, physiological and biochemical changes. Alterations include changes in the biosynthetic activity and release of macromolecules by the oviductal epithelium which become part of the luminal microenvironment. Furthermore, both regional and temporal differences in activity and protein production occur through hormonal changes during the oestrous cycle and early pregnancy. Studies on identification, characterization and regulation of several proteins synthesized de novo have indicated oocyte-oviduct and embryo-oviduct interactions. However, the identification of oviduct-derived proteins, their regulation and their potential function in vivo needs to be examined. Studies in other species also suggest roles for growth factors in early embryonic development, but little information is available for the pig. We propose that ovarian hormones control changes in synthetic activity, synthesis of some oviduct-derived proteins and the presence of specific factors in the luminal microenvironment which sustain and enhance fertilization and early cleavage-stage embryonic development.