Preimplantation factor negates embryo toxicity and promotes embryo development in culture.

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients (n=14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14-3.55, chi-squared=12.74, P=0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11-0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared=4.0, P=0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models.

BACKGROUND: PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. METHODS: Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. RESULTS: PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). CONCLUSIONS: PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.

Ooplasm transfer and interspecies somatic cell nuclear transfer: heteroplasmy, pattern of mitochondrial migration and effect on embryo development.

Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

Isolation and culture of porcine adipose tissue-derived somatic stem cells.

Adipose tissue-derived stem cells (ASCs) have been described for a number of laboratory animals and humans. Improved culture conditions and cellular characteristics of ASCs have been identified. ASCs can self-renew and differentiate into multiple tissue lineages. Further characterization of ASCs in this manner could enhance the isolation and purification of a population of mesenchymal stem cells (MSCs) from easily obtainable adipose tissue. These stem cell populations from domestic animals, which make attractive models for transplantation studies, will be valuable for the evaluation of their efficacy in tissue regeneration applications in the future. These cells may also represent a population more easily reprogrammable during somatic cell nuclear transfer and thus expedite the development of transgenic animals for models and production of valuable pharmaceutical proteins.

Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer.

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the DeltaDeltaC(T) method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.

Sex ratio of bovine embryos and calves originating from the left and right ovaries.

An asymmetric distribution of the sexes within the left and right uterine horns has been described in multiple species. A series of experiments were conducted to evaluate the sex ratio (% male) of calves gestated in the left and right uterine horns, as well as the sex ratio of embryos originating from the left and right ovaries of cattle. The sex ratio of calves gestated in the right uterine horn of naturally mated cows was significantly higher compared with the sex ratio of calves gestated in the left uterine horn. In addition, the sex ratio of the left and right uterine horns differed significantly from parity. The sex ratio of embryo transfer calves born following transfer to the left and right uterine horns was not significantly different. Additionally, the proportion of male embryos collected from the right uterine horns was significantly greater than from the left uterine horns of superovulated cows. The sex ratio of embryos collected from the left and right uterine horns of unilaterally ovariectomized cows was not significantly different. However, more female than male embryos were produced when left ovary oocytes fertilized in vitro. In conclusion, the results of these experiments demonstrate that a significantly greater proportion of males are gestated in the right uterine horn of cattle and a greater proportion of females in the left. Additionally, the data indicate that sex-specific selection pressure may be applied to embryos by ovarian factors rather than by the uterine environment.

Ultrasonographic-guided retrieval and in vitro maturation of eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus isaaci) antelope oocytes.

The limited availability of gametes is a major factor hindering the development and application of assisted reproductive technologies (ART) in large non-domestic ungulates. This is partly due to the small number of captive animals and handling difficulties associated with procedures for gamete recovery. In the present study, results are reported of multi-year studies on ovarian stimulation and oocyte retrieval by ultrasonographic-guided transvaginal follicular aspiration and subsequent in vitro maturation (IVM) in eland and bongo antelopes. All procedures were conducted on sedated females handled in a hydraulic chute without inducing general anesthesia. Five estrous synchronization/ovarian stimulation protocols were evaluated and data are presented on 73 and 15 procedures in eland and bongo, respectively. Repeating procedures (< or =once/month) on the same female did not affect ovarian response or number oocytes recovered in either species. Eland females, but not the ovarian stimulation treatment, affected ovarian response. Ovarian stimulation treatment affected oocyte recovery rate in eland, but not in bongo. In both species, ovarian hormone stimulation treatment affected the distribution of follicles by size and the status of expansion of the cumulus cell investment of oocytes, but not the frequency of metaphase II oocytes during IVM. The timing of extrusion of the first polar body during IVM was more synchronous in bongo than in eland oocytes. It is concluded that Transvaginal oocyte retrieval (TVOR) can be safely and repeatedly applied in gonadotropin-treated eland and bongo females to recover oocytes that can mature in vitro. The methods described for the present study can be adapted to improve the availability of non-domestic ungulate oocytes for basic and applied studies.

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

BACKGROUND: Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. METHODS: Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. RESULTS: An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. CONCLUSION: Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34.

Cloned embryos from semen. Part 2: intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes.

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had > or = 8 cells at 84 hpa, while 32% of the bovine NT embryos had > or = 8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had > or = 8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further.

Cloned embryos from semen. Part 1: in vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation.

Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

Effect of epigenetic modifications of donor somatic cells on the subsequent chromatin remodeling of cloned bovine embryos.

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.

Reversal of motility loss in bongo antelope (Tragelaphus eurycerus isaaci) spermatozoa contaminated with hyposmotic urine during electroejaculation.

Semen collected by a combination of ampullary (rectal) massage and electroejaculation of a bongo bull was incidentally contaminated with urine (1:3.7). At 1.5h post-collection, progressive motility was 0% but some spermatozoa had intermittently twitching tails. Subsequent dilution with media and processing improved the progressive motility (up to 50%) and intact membranes (up to 71%) of spermatozoa. After thawing, the respective values were 35 and 70%. The osmolarity and pH of the contaminated supernatant was 151 mOsm and 7.45, respectively. Initial progressive motility in a non-contaminated portion of semen collected during the same procedure was 80%, and, after thawing, 60 and 90%, of the spermatozoa showed progressive motility and intact membranes, respectively. In conclusion, urine-contaminated bongo spermatozoa can regain progressive motility after dilution with isosmotic solutions and survive cryopreservation.

DNA methylation and histone acetylation patterns in cultured bovine fibroblasts for nuclear transfer.

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of a differentiated cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst production and development to term of cloned embryos may differ between population doublings (PDs) of the same cell line as a consequence of changes in DNA methylation and histone acetylation patterns during in vitro culture. The objective of this study was to determine gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in addition, to measuring levels of DNA methylation and histone acetylation of bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, counter-stained with propidium iodide and analyzed by flow cytometry. These data demonstrate that chromatin remodeling protein mRNAs involved in epigenetic modifications are altered during in vitro culture. Methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.

Proliferative characteristics and chromosomal stability of bovine donor cells for nuclear transfer.

Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. Abnormal phosphorylation patterns of the histones during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine proliferative characteristics, chromosomal stability, and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation. Proliferative characteristics, percentage of aneuploid cells, and relative levels of phosphorylated histone H3 (ser10) were determined at different population doublings (PD) by cell counting, karyotyping, and flow cytometry, respectively. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. Some cell lines had up to 50% of aneuploid cells during early passages. Multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture (60 and 41%, respectively). An increase in the relative level of phosphorylated histone occurred after extended time in culture (55.7 during early passages vs. 102.6 at late passages). These data demonstrate the importance of determining chromosome content and the selection of healthy cell lines to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of nuclear transfer (NT).

Behavioral training and hydraulic chute restraint enables handling of eland antelope (Taurotragus oryx) without general anesthesia.

Difficulties and risks associated with restraining large nondomestic ungulates are limiting factors toward developing and applying assisted reproductive technologies, such as artificial insemination and embryo transfer. In this study on 10 female eland (Taurotragus oryx), we evaluated the use of behavioral training and handling handling in a hydraulic chute to perform transvaginal ultrasound-guided oocyte retrieval and other clinical procedures. Nine females were conditioned to associate specific sound cues with food treats. The interval from the audio cue until acceptance of handheld treats varied among females (1.8-58.3 min). Animals also differed in their response to training for voluntary entry into the chute. Handling eland for oocyte retrieval in the hydraulic chute required xylazine sedation. During sedation and handling, eland undergoing oocyte retrieval procedures had higher blood glucose levels (14.4 +/- 3.1) than females handled similarly but without oocyte retrieval (9.3 +/- 2.7 mmol/L). Plasma osmotic pressure, hematocrit, and creatine phosphokinase activity were similar between these two groups. Females that were more difficult to train had higher blood glucose levels than the more cooperative animals. Cooperative females had fewer vertical stripes on their sides. More than 40 procedures were conducted without complications or mortality. The combination of behavioral conditioning-training and restraint of sedated eland in a hydraulic chute was a reliable and repeatable method for performing minimally invasive assisted reproductive techniques.

Endocrine profiles and growth patterns of cloned goats.

Two groups of goats produced by fetal somatic cell nuclear transfer (NT) were monitored to evaluate the similarities in growth patterns among cloned animals. Clone group I consisted of five Toggenburg females cloned from the same transgenic cell line and born to different recipient does. Clone group II consisted of two Saanen does born as twins to a single recipient female from a second transgenic cell line. Each cell line was constructed with a transgene (different for each clone group) that would express, producing a protein product in the milk during lactation of the does. Weight, hip height, and circulating levels of growth-related hormones were monitored at weekly and monthly intervals for comparison within the clone groups. A contemporary group (group III) consisting of seven crossbred does of similar ages and weights was also monitored for baseline endocrine values during the study. Serum samples from all groups were analyzed for growth hormone (GH), insulin-like growth factor I (IGF-I), triiodothyronine (T3), thyroxine (T4), and insulin via standard laboratory radioimmunoassay procedures. The averaged standard deviation from the mean was used to evaluate similarities within the groups of cloned does and the does in the contemporary group. The does in clone group II were less variable than the goats in clone group I for weight and hip height. This was perhaps due to a recipient effect. The two groups of cloned females were less variable than the contemporary group for circulating IGF-I and T4 concentrations. In contrast, the two groups of cloned does had at least one cloned group that was more variable than the contemporary group for GH, T3, and insulin. The animal variation, measured by the average standard deviation from the mean, of the cloned does is possibly due to environmental effects encountered in utero and/or in the postnatal period, as well as possible mitochondrial DNA differences between the cell line and donor oocytes used for NT. The variation among cloned does in this study indicates that the use of somatic cell NT to reproduce identical phenotypes may not succeed in all situations.

Birth of African Wildcat cloned kittens born from domestic cats.

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.

Production of nuclear transfer llama (lama glama) embryos from in vitro matured llama oocytes.

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p < 0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.