Characterisation of sperm production and morphology in the male Philippine crocodile Crocodylus mindorensis via voluntary behavioural training.

Philippine crocodiles Crocodylus mindorensis are critically endangered due to agricultural and fishing threats that have severely fragmented their habitat and population in the Philippines. Captive management plans are important to safeguard against their extinction, but the current population in US zoos is small, and breeding is hampered by the slow growth of this species and the danger of introducing differently sized animals for breeding. There is little information regarding the sperm characteristics of crocodilians, and none for Philippine crocodiles. In this study, we sought to characterise sperm production in the male Philippine crocodile (n =1) by performing voluntary (without sedation or restraint) collections (n =181) over a 3.5-year period. Peak sperm production in this individual occurs from January to July, when the mean (±s.e.m.) total number of spermatozoa recovered was 10.2×106 ±3.8×106 (n =104), compared with 0.3×106 ±0.2×106 (n =71) for all other months of the year. Analysis of sperm morphology indicated that 15.9% of spermatozoa exhibited normal morphology. A bent tail was the most common abnormality (48.2%) observed. Understanding the basic reproductive biology of the male Philippine crocodile will facilitate the development of artificial reproductive technologies to improve captive propagation and genetic management of this species.

Assisted reproductive technologies for endangered species conservation: developing sophisticated protocols with limited access to animals with unique reproductive mechanisms.

Assisted reproductive technologies (ARTs) have been proposed as a means of overcoming the significant challenges of managing small, isolated populations of endangered species in zoos. However, efficient protocols for ARTs do not exist for most endangered species. This review will focus on research efforts to characterize unique reproductive mechanisms and develop species-specific ARTs. Central to these studies are assays to measure steroid metabolites in urine or feces and/or training programs to allow unrestrained blood collections and ultrasound evaluations. The resulting information about estrous cycle dynamics, combined with studies of semen collection and processing, provides the foundation for the development of artificial insemination (AI). In vitro fertilization and embryo transfer are also discussed in relation to the advantages these techniques could provide relative to AI, as well as the significant challenges involved with technologies that require oocytes and embryos. Finally, an argument is made for additional research of nontraditional model species (e.g., domestic cats and dogs) and the development of novel models representing unique taxa. Whether these species are studied by zoo-based researchers with the expressed intent of developing ARTs for conservation or academic scientists interested in basic biology, the resulting information will provide a unique, evolutionary perspective on reproduction that could have wide-reaching benefits. The more information we have available, the better our chances will be of developing effective ARTs and making a difference in conservation efforts for endangered species.

A carnivore embryo's perspective on essential amino acids and ammonium in culture medium: effects on the development of feline embryos.

Carnivores are an interesting model for studies of embryonic amino acid metabolism and ammonium (NH4+) toxicity given the high-protein content of their diets. Our objectives were to examine concentration- and stage-specific effects of essential amino acids (EAA; 0×, 0.125×, 0.25×, 0.5×, or 1.0× the concentrations in Minimum Essential Medium) and NH4+ (0, 300, or 600 µM) on the development and metabolism of feline embryos. The presence of EAA, regardless of concentration, during days 3-7 of culture increased (P < 0.01) the proportion of embryos that initiated hatching (>14.3%) and the total number of cells per blastocyst (>148.3 cells) compared to embryos cultured without EAA (0.0% and 113.2 ± 3.7 cells, respectively). The presence of EAA during days 1-3 (0.25×) and 3-7 (1.0×) of culture increased (P < 0.01) the proportions of embryos that formed blastocysts (82.9 ± 4.2%) and initiated hatching (32.9 ± 5.2%), and the number of cells per blastocyst (247.9 ± 12.1 cells), compared to control embryos (60.0 ± 5.3%, 0.0%, 123.2 ± 8.1 cells, respectively). The presence of NH4+ in the medium did not affect (P > 0.05) development of feline embryos. The addition of EAA or NH4+ during culture did not affect (P > 0.05) the production of Gln by feline embryos, but decreased (P < 0.05) production of Ala and increased (P < 0.05) production of urea. Additional work is needed to determine if our observations are unique to feline embryos or reflect an adaptation to a high-protein diet that is conserved in other carnivores.

Exogenous growth factors do not affect the development of individually cultured murine embryos.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 µl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P < 0.05) effect of growth factors was a decrease in the proportion of embryos that formed blastocysts by day 5 in CF1 IVM embryos. The presence of growth factors also did not affect blastocyst cell numbers. For CF1 IVO embryos, the presence of growth factors during culture did not affect the proportion of embryos that attached to fibronectin-coated dishes, the size of the resulting outgrowths, or in vivo development following transfer. CONCLUSION: Combinations of paf, EGF, GM-CSF, and IGF-1 did not improve development of murine embryos cultured individually in a sequential medium containing a defined protein source.

Transporting cumulus complexes using novel meiotic arresting conditions permits maintenance of oocyte developmental competence.

PURPOSE: The aim of this study is to evaluate the effect of a novel bovine cumulus oocyte complex (COC) shipping media designed to arrest meiotic resumption during transport on meiotic arrest, as well as meiotic resumption, subsequent embryonic development, and embryo quality. METHODS: Bovine cumulus oocyte complexes were transported overnight from the collection facility to the laboratory. COCs were placed in control in vitro maturation (IVM) or in shipping arrest medium (SAM) containing multiple meiotic inhibitors, and then shipped to our laboratory. Upon arrival, meiotic status was assessed, control COCs were inseminated, and arrested COCs were matured and inseminated the next day. Embryonic development and quality were analyzed. RESULTS: When bovine COC arrived at the laboratory after overnight shipment (21 h) in SAM, the majority of oocytes remained at the GV stage (75.6 ± 2.9% GV). Arrested oocytes successfully resumed and completed meiosis during IVM after removal from SAM (96.8 ± 0.5% metaphase II compared to control 88.3 ± 5.0%). Moreover, the development of blastocysts per COC was not different from control (22.3 ± 2.4% for control and 18.7 ± 2.1% for SAM), nor was any difference detected in blastocyst quality as determined by cell number and allocation. CONCLUSIONS: Our study demonstrates that a physiological system incorporating cyclic adenosine monophosphate and cyclic guanosine monophosphate modulators can be used to maintain meiotic arrest followed by successful nuclear maturation and pre-implantation embryo development equal to control IVM-derived embryos. Our results offer promising insights for the development of pre-IVM media that may improve oocyte developmental competence in vitro.

The effects of permeating cryoprotectants on intracellular free-calcium concentrations and developmental potential of in vitro-matured feline oocytes.

Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca(2+)]i), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca(2+)]i, but changes in [Ca(2+)]i, resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (<5% for all treatments) were not affected (P>0.05) by extracellular Ca(2+). Exposure to EG (>44.1%) and DMSO (19.7%) increased (P<0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (≤2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P>0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage.

Building a better mouse embryo assay: effects of mouse strain and in vitro maturation on sensitivity to contaminants of the culture environment.

PURPOSE: The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice. METHODS: The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 % v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated. RESULTS: Neither concentration of TX100 affected (P > 0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P < 0.05) by the high concentration of TX100 and the number of ICM cells was reduced (P < 0.05) by the low concentration of TX100. Inhibitory (P < 0.05) effects of the high concentration of TX100 were observed in in vitro maturation (IVM) embryos from BDF1, CF1, and SW, but not ICR, mice. Cell number and allocation were negatively affected by the high concentration of TX100 in CF1 and SW embryos, but not in BDF1 or ICR embryos. The only developmental endpoints affected by the low concentration of TX100 were cleavage of BDF1 oocytes, blastocyst development of SW embryos, and cell numbers (total and inner cell mass (ICM)) of SW blastocysts. CONCLUSIONS: The sensitivity of the MEA to TX100 is improved by using embryos from in vitro-matured oocytes, using oocytes from some outbred (SW or CF1, not ICR) strains of mice, and evaluating blastocyst cell number and allocation.

Antioxidant supplementation during in vitro culture improves mitochondrial function and development of embryos from aged female mice.

Maternal aging results in reduced oocyte and blastocyst quality, thought to be due, in part, to mitochondrial dysfunction and accumulation of reactive oxygen species. To reduce oxidative stress, the antioxidants α-lipoic acid (ALA; 10µM), α-tocopherol (250µM), hypotaurine (1mM) and N-acetylcysteine (NAC; 1mM), and sirtuin (100ngmL(-1)) were added to embryo culture medium (AntiOX) and compared with a control (CON) without antioxidants to assess blastocyst development after in vitro maturation and fertilisation of oocytes from aged B6D2F1 female mice (13.5 months). Development to the blastocyst stage increased in the AntiOX compared with CON group (87.6% vs 72.7%, respectively; P<0.01), in addition to higher mitochondrial membrane potential and ATP levels in the AntiOX group. Expression of genes associated with oxidative stress (PI3K, FOXO3A and GLRX2) was upregulated in the CON compared with AntiOX group. In addition to AntiOX, a medium containing only NAC and ALA (rAntiOX) was used to culture embryos from young CF1 females (6-8 weeks). More blastocysts developed in the rAntiOX compared with CON group (64.1% vs 43.3%, respectively; P<0.01), although AntiOX (48.0% blastocysts) did not result in improved development in young mice. Antioxidants improved mitochondrial activity, gene expression and development in embryos of older female mice, whereas a reduced level of antioxidants during culture was beneficial to embryos from young mice.

The beneficial effects of reduced magnesium during the oocyte-to-embryo transition are conserved in mice, domestic cats and humans.

In many cell types Mg2+ can antagonise Ca2+ -stimulated signalling pathways, but information regarding the effects of these ions on IVF and subsequent embryonic development is limited. Our objectives were to evaluate the effects of Mg2+ in the IVF medium on embryonic development in mice and then determine if similar effects occurred in domestic cats and humans. Oocytes from hybrid and outbred mice, domestic cats and humans were fertilised (IVF, mice and cats; intracytoplasmic sperm injection (ICSI), humans) in the presence of 0.2 or 1.2 (mouse and human) or 1.0 (cat) mM Mg2+ and the resulting embryos were cultured to the blastocyst stage. Decreased concentrations of Mg2+ during IVF increased (P<0.05) cleavage of oocytes from outbred mice (77.9 vs. 51.0%), development of embryos from hybrid mice (74.5 vs. 51.0% hatching blastocyst per cleaved embryo) and both cleavage (68.4 vs. 46.8%) and blastocyst development (53.0 vs. 26.2% per cleaved embryo) in cats. Development to the blastocyst stage (52.1 vs. 40.2%) was also improved (P<0.05) when ICSI was performed on human oocytes in the presence of 0.2 mM Mg2+, compared with a commercial culture medium. Sensitivity to increased (1.0 to 1.2 mM) concentrations of Mg2+ in the medium during the oocyte-to-embryo transition appears to be conserved in three different species.

Applying metabolomic analyses to the practice of embryology: physiology, development and assisted reproductive technology.

The advent of metabolomics technology and its application to small samples has allowed us to non-invasively monitor the metabolic activity of embryos in a complex culture environment. The aim of this study was to apply metabolomics technology to the analysis of individual embryos from several species during in vitro development to gain an insight into the metabolomics pathways used by embryos and their relationship with embryo quality. Alanine is produced by both in vivo- and in vitro-derived human, murine, bovine and porcine embryos. Glutamine is also produced by the embryos of these four species, but only those produced in vitro. Across species, blastocysts significantly consumed amino acids from the culture medium, whereas glucose was not significantly taken up. There are significant differences in the metabolic profile of in vivo- compared with in vitro-produced embryos at the blastocyst stage. For example, in vitro-produced murine embryos consume arginine, asparagine, glutamate and proline, whereas in vivo-produced embryos do not. Human embryos produce more alanine, glutamate and glutamine, and consume less pyruvate, at the blastocyst compared with cleavage stages. Glucose was consumed by human blastocysts, but not at a high enough level to reach significance. Consumption of tyrosine by cleavage stage human embryos is indicative of blastocyst development, although tyrosine consumption is not predictive of blastocyst quality. Similarly, although in vivo-produced murine blastocysts consumed less aspartate, lactate, taurine and tyrosine than those produced in vitro, consumption of these four amino acids by in vitro-derived embryos with high octamer-binding transcription factor 4 (Oct4) expression, indicative of high quality, did not differ from those with low Oct4 expression. Further application of metabolomic technologies to studies of the consumption and/or production of metabolites from individual embryos in a complete culture medium could transform our understanding of embryo physiology and improve our ability to produce developmentally competent embryos in vitro.

Reversible meiotic arrest in feline oocytes.

Increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP) within the cumulus-oocyte complex (COC) inhibits or delays spontaneous oocyte maturation and improves the developmental competence of the oocyte in many species, but information for carnivores is limited. The objectives of the present study were to describe the effects of isobutyl methylxanthine (IBMX), which decreases cAMP degradation, and forskolin, which increases cAMP production, on spontaneous and induced maturation (by equine chorionic gonadotrophin (eCG) and epidermal growth factor (EGF)) of feline oocytes and to evaluate the reversibility of IBMX-induced arrest by measuring the resumption of meiosis and embryonic development following IVF. IBMX decreased (P<0.05) the incidence of spontaneous (6.7% vs 42.0%, metaphase II (MII)) and induced (5.6% vs 66.1% MII) maturation after 24 h of culture. In contrast, forskolin stimulated meiosis (81.7% MII; P<0.05). Following 12 h of culture with IBMX and an additional 24h with eCG and EGF in the absence of IBMX, the proportions of oocytes reaching MII (66.1%), cleaving (79.9%) and developing to the blastocyst stage (15.3%) were similar (P>0.05) to oocytes cultured continuously with eCG and EGF (70.2%, 83.0% and 18.1%, respectively). These results demonstrate that IBMX reversibly inhibits both spontaneous and eCG+EGF-induced meiosis in feline oocytes without compromising the oocyte's developmental competence.

Lipid Enriched Reduced Nutrient Culture Medium Improves Bovine Blastocyst Formation.

The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.

Nutrient intake and influence on markers of oxidative stress in zoo-managed male snow leopards (Uncia uncia).

Oxidative stress (OS) results from the overproduction of reactive species. Nutrient intake can contribute positively or negatively to OS, and the lack of established nutrient requirements for most of the exotic species managed in zoos exacerbates the possibilities for nutrient imbalances that potentially could lead to reactive species production. The objective of this study was to evaluate the influence of nutrient intake and nutritional husbandry on markers of OS in male snow leopards (n = 14) maintained in U.S. facilities (n = 12). Diet samples and husbandry information were obtained and snow leopards were immobilized once for collection of blood. Samples were analyzed for chemical composition (diet and blood), antioxidant capacity (blood), and markers of OS (blood). Correlations between weekly nutrient intakes and markers of OS were analyzed by linear regression. Analyzed markers of OS included antioxidant enzymes (superoxide dismutase [SOD] and glutathione peroxidase [GPx]) and ferric reducing antioxidant potential that are protective against OS, and protein carbonyls, thiobarbituric acid reactive substances, and DNA/RNA damage that are indicative of oxidative damage. Weekly copper intake (10.1 to 80.2 mg) was negatively correlated with DNA/RNA damage (R 2 = 0.44; P = 0.01). Weekly sodium intake (4.4 to 12.7 g) was positively correlated with GPx activity (R 2 = 0.43; P = 0.04). More frequent feeding of whole prey (0.3 to 3 times/wk) was correlated with increased blood SOD activity (R 2 = 0.55; P < 0.01). In conclusion, greater dietary copper intake and more frequent feeding of whole prey may reduce OS in snow leopards. Dietary sodium intake and relationship with GPx activity should be further evaluated to determine benefit or detriment. No cause and effect can be inferred from our results, but our data suggest altering dietary form and nutrient concentrations may influence OS in snow leopards.

Fatty acids present in commercial albumin preparations differentially affect development of murine embryos before and during implantation.

OBJECTIVE: To characterize fatty acid (FA) profile of commercially available albumin products and determine their effect on embryonic development. DESIGN: Research study. SETTING: Private research facility. ANIMAL(S): Outbred mice aged 4-8 weeks. INTERVENTION(S): Gas chromatography-mass spectrometry was used to quantify the FA content of 15 commercial albumins. Embryos were produced in media containing different albumin products, with or without carnitine or exogenous FA supplementation, to determine their effect on embryo development in vitro. MAIN OUTCOME MEASURE(S): Total micrograms of FA per milligram of albumin for the 15 albumin products, blastocyst development, cell number, allocation to the trophectoderm (TE) or inner cell mass (ICM), and evaluation of morphology during implantation. RESULT(S): The albumin products contained 0.07-16.77 µg total FA/mg albumin. Compared to media with with >1.4 µg FA/mg albumin, media with <0.5 µg FA/mg albumin supported improved blastocyst development, and addition of carnitine mitigated this difference. Addition of palmitoleic acid or oleic acid individually did not improve blastocyst development and decreased ICM:TE ratio. However, in the presence of carnitine, there was improved blastocyst development and maintenance of the ICM:TE ratio. Embryos cultured in Vitrolife human serum albumin with supplementation of carnitine, palmitoleic acid, and oleic acid were more likely to develop cells positive for POU5F1 in an extended embryo culture than embryos cultured in Origio serum protein substitute. CONCLUSION(S): Commercial albumin products contain FAs, which vary in abundance. These FAs have different effects on embryo development and quality before and during the implantation stage. Several of these albumin preparations are routinely used for human-assisted reproductive technologies; therefore, serious consideration is warranted when selecting a product for clinical use.

Cross-species efficacy of a chemically-defined, soy lecithin-based cryomedium for semen banking in imperiled wild felids.

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium. However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. In the present study, our aim was to compare a chemically-defined, soy-based medium (SOY) to a commercial egg yolk-based medium (TEY) for the cryopreservation of sperm in four imperiled small cat species. Semen was collected from adult male cats (n = 6 black-footed cats; n = 6 sand cats; n = 4 fishing cats; and n = 7 Pallas' cats) via electroejaculation, split into two aliquots, and cryopreserved in SOY or TEY. Frozen-thawed samples were evaluated for sperm motility and rate of progressive motility (up to 24 h post-thaw) and acrosome status (0 and 6 h). No difference in post-thaw traits were observed between treatments in all four species. Heterologous IVF using oocytes collected laparoscopically from domestic cats demonstrated no difference among freezing treatments in percentage of mature oocytes that cleaved or the mean number of blastomeres at 48 h post-insemination. More spermatozoa frozen with SOY were bound to the zona pellucida in the sand cat (P = 0.018), but no treatment effect was observed in the other three species. These findings collectively demonstrate that SOY may be a preferable alternative to TEY for sperm cryopreservation in these four wild felid species.

The landscape of accessible chromatin in bovine oocytes and early embryos.

Chromatin reorganization governs the regulation of gene expression during preimplantation development. However, the landscape of chromatin dynamics in this period has not been explored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) which revealed unique features of the accessible chromatin during bovine early embryo development. We found that chromatin accessibility is low in oocytes and 2-/4-cell embryos, followed by a significant increase in embryos during major embryonic genome activation (EGA), and peaked in elongating day 14 embryos. Genome-wide characteristics of open chromatin showed that ATAC-seq signals in both transcription start sites (TSS) and transcription end sites (TES) were strong. Additionally, the distal ATAC-seq peaks were enriched in repeat elements in a type-specific and stage-specific manner. We further unveiled a series of transcription factor (TF) motifs with distinct variation of enrichment from distal ATAC-seq peaks. By integrated analysis of chromatin accessibility with transcriptomes and DNA methylomes in bovine early embryos, we showed that promoter accessibility was positively correlated with gene expression, especially during major EGA, and was strongly correlated to DNA methylation and CpG density. Finally, we identified the critical chromatin signatures and TFs that differ between in vivo and in vitro derived blastocysts, which provides insights to the potential mechanisms leading to low quality of embryos produced in vitro. Together, this comprehensive analysis revealed critical features of chromatin landscape and epigenetic reprogramming during bovine preimplantation embryo development.

Factors affecting reproductive traits in male snow leopards (Unciauncia).

ABSTRACT: The population of snow leopards (Unciauncia) maintained in US zoos is no longer sustainable due to poor reproductive success. Our objective was to assess reproductive traits in male snow leopards and identify factors (markers of oxidative stress in seminal fluid, surveys of husbandry practices, gonadal and adrenocortical activity, dietary intake of various nutrients, and genetics) that may affect ejaculate traits and subsequent fertility. Ejaculates (2.9 ± 0.2 mL) from 32 male snow leopards (9.8 ± 0.7 years; 38.6 ± 0.8 kg) housed at 27 institutions contained 119.2 + 26.0 x 106 spermatozoa, of which 75.1 ± 2.3% were motile and 28.6 ± 2.6% exhibited normal morphology. Overall, 34% of males produced <5 million spermatozoa and 27% of males produced spermatozoa with <20% normal morphology. Activity of superoxide dismutase (SOD) in the seminal fluid was negatively correlated (P < 0.05, r2 = 0.90) with normal sperm morphology. Husbandry practices, mean concentrations of fecal androgen metabolites (fAM), and baseline concentrations of fecal glucocorticoid metabolites (fGM), inbreeding coefficients, and generations each male was removed from the founders in their lineages were not correlated (P > 0.05) with the total number of spermatozoa or the proportion of spermatozoa with normal morphology. Total sperm count was positively correlated (P < 0.05, R2 = 0.86) with the weekly intake of polyunsaturated fatty acids (PUFA) and the proportion of spermatozoa with normal morphology tended (P < 0.10, R2 = 0.31) to be positively correlated with copper intake. Altering the nutrient composition of snow leopard diets could provide managers with a possible method of improving reproductive traits in this endangered species. LAY SUMMARY: The population of snow leopards (Uncia uncia) maintained in US zoos has been declining since 1993 due to poor breeding success. Our objective was to assess the reproductive traits of male snow leopards and identify factors (e.g. hormones, diet, genetics) that may be affecting the quality of semen produced and therefore subsequent fertility. Within a cohort of 32 male snow leopards maintained at 27 US zoos, we found that 34% produced less than 5 million sperm and 27% of males produced sperm where less than 20% looked normal. The quantity and quality of the recovered sperm was not correlated with husbandry practices, concentrations of hormones (androgens and glucocorticoids) in feces, or genetics. However, the number of sperm was positively correlated with polyunsaturated fatty acids in the diet. Altering the nutrient composition of snow leopard diets could provide managers with a possible method of improving reproductive traits in this endangered species.

Developmental and molecular response of bovine embryos to reduced nutrients in vitro.

Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8-124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media. LAY SUMMARY: To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

A novel culture medium with reduced nutrient concentrations supports the development and viability of mouse embryos.

Further refinement of culture media is needed to improve the quality of embryos generated in vitro. Previous results from our laboratory demonstrated that uptake of nutrients by the embryo is significantly less than what is supplied in traditional culture media. Our objective was to determine the impact of reduced nutrient concentrations in culture medium on mouse embryo development, metabolism, and quality as a possible platform for next generation medium formulation. Concentrations of carbohydrates, amino acids, and vitamins could be reduced by 50% with no detrimental effects, but blastocyst development was impaired at 25% of standard nutrient provision (reduced nutrient medium; RN). Addition of pyruvate and L-lactate (+PL) to RN at 50% of standard concentrations restored blastocyst development, hatching, and cell number. In addition, blastocysts produced in RN + PL contained more ICM cells and ATP than blastocysts cultured in our control (100% nutrient) medium; however, metabolic activity was altered. Similarly, embryos produced in the RN medium with elevated (50% control) concentrations of pyruvate and lactate in the first step medium and EAA and Glu in the second step medium were competent to implant and develop into fetuses at a similar rate as embryos produced in the control medium. This novel approach to culture medium formulation could help define the optimal nutrient requirements of embryos in culture and provide a means of shifting metabolic activity towards the utilization of specific metabolic pathways that may be beneficial for embryo viability.

Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer.

BACKGROUND: Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer. METHODS: First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B) to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats. RESULTS: All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220) were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred. CONCLUSION: Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations.