Effects of freeze-drying on the quality and fertilising ability of goat sperm recovered from different parts of the epididymis.

Lyophilisation is an alternative method for sperm preservation. The aim of this study was to evaluate the effects of freeze-thawing (F/T) and freeze-drying (F/D) on the quality of epididymal goat sperm. Sperm from each region of the epididymis (caput, corpus and cauda) were collected and evaluated for the expression of phospholipase C zeta (PLC-ζ), protamine 1 (PRM1), transition protein 1 (TNP1) and 2 (TNP2). The effects of F/T and F/D on sperm quality in terms of PLC-ζ expression, chromatin stability (Chromomycin A3; CMA3) and DNA integrity were examined. The fertilising ability after intracytoplasmic sperm injection (ICSI) was also tested. Fresh sperm existed PLC-ζ, PRM1, TNP1 and TNP2, irrespective of the regions of the epididymis. However, different patterns of PLC-ζ expression were found. Although PRM1, TNP1, TNP2 were still expressed after F/T or F/D, only F/T could preserve the presence of PLC-ζ. For fresh sperm, caput epididymal sperm had the lowest evidence of chromatin stability when compared to sperm harvested from other regions of the epididymis. The F/T and F/D further increased the numbers of CMA3-positive sperm (P < 0.001). In all cases, no CMA3 staining was observed in caudal epididymal sperm. The caudal epididymal sperm had significantly greater proportions of sperm with intact DNA compared with caput and corpus epididymal sperm, especially when F/T and F/D were performed. The fertilisation rates of F/D sperm tended to decrease when compared with F/T sperm (4.2 ± 3.2 vs. 13.6 ± 9.0, P = 0.08). It is concluded that the sperm recovered from the caudal epididymis is suitable for freezing and lyophilisation. However, poor fertilisation rates of F/D sperm were coincidently observed, with a deficit demonstration of PLC-ζ.

In Vitro Effects of Short-Term and Long-Term Heat Exposures on the Immune Response and Prostaglandin Biosynthesis in Bovine Endometrial Cells.

Worldwide heat stress (HS) conditions have a negative impact on dairy cow fertility. However, understanding of the effect of heat stress on endometrial functions is still unclear. The present study aimed to investigate the effects of differential heat exposure conditions on the immune response and prostaglandin biosynthesis of bovine endometrium challenged with bacterial lipopolysaccharide (LPS). Cultures of endometrial cells were grown to confluence at 37 °C (control) and 40.4 °C for 24 h after confluence (short-term heat exposure) and 40.4 °C for 8 days from the beginning of the culture (long-term heat exposure), prior to a challenge by 100 ng/mL LPS for 12 h. LPS altered ALOX12, IL8, IL1B, S100A8, PTGES and AKR1B1 expressions, as well as secretory IL8 and PGF2α. Short-term heat exposure decreased S100A8, IL8 and PGF2α compared with the control temperature, while long-term heat exposure decreased S100A8 and PGF2α. In contrast, HSPA5 expression was not altered by heat exposure or LPS. Indeed, the short-term heat treatment was insufficient for accomplishing the responses of the endometrium to LPS treatment for IL8, S100A8 and PTGES expressions when compared with other temperature conditions. Our findings showed that heat exposure could compromise endometrium immune response and prostaglandin biosynthesis in different ways based on elevated temperature duration, which could reduce subsequent fertility.

Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility.

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.

CRISP protein expression in semen of the endangered Malayan tapir (Tapirus indicus).

The Malayan tapir is a large endangered herbivore native to South-east Asia with fewer than 2500 animals remaining in the wild. Although a small number of animals (183 animals held by 60 institutions) are managed in zoos and breeding centres, there is limited information on the fundamental reproductive biology of this species. The purpose of this present study was to evaluate the associations of reproductive protein biomarkers (CRISP2 and CRISP3) in the seminal plasma and spermatozoa with reproductive characteristics in male Malayan tapirs. Ejaculates were collected from zoo-housed animals by electroejaculation and assessed for sperm motility and quality traits. Seminal plasma and sperm pellets were analysed for CRISP protein expression by immunoblotting. The reproductive tract of a single animal was also analysed for CRISP2 and CRISP3 protein expression and localization by immunohistochemistry. Our results showed that both CRISP2 and CRISP3 are expressed in the seminal plasma and spermatozoa derived from Malayan tapirs. CRISP expression was positively correlated with semen quality, especially ejaculate volume, number of motile sperm, and acrosomal integrity. In addition, CRISP2 and CRISP3 protein expression were slightly high in males that had recently sired an offspring. The results suggest that CRISP proteins may serve as biomarkers for ejaculate quality and fertility in male Malayan tapirs. These findings may have significant implications for planning future breeding and re-introduction efforts for this species.

Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells.

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.

Generation of porcine induced-pluripotent stem cells from Sertoli cells.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming of somatic cells using four transcription factors: OCT4, SOX2, KLF-4, and c-MYC (OSKM). However, reprogramming efficiency of iPSCs is currently poor. In this study, we used the Sertoli line as a novel cell source for somatic cell reprogramming. Neonatal testes were collected from 1-week-old piglets. The testes were digested by a two-step enzymatic method to isolate Sertoli cells. The latter were transfected with retroviral vectors expressing OSKM. The Sertoli iPSC-like colonies were subjected to morphological analysis, alkaline phosphatase staining, RT-PCR, G-banding karyotyping, in vitro differentiation, and in vivo differentiation. Primary Sertoli cells had polygon-shaped morphology and manifested phagocytic activity as determined by a fluorescent bead assay. Sertoli cells also expressed the anti-Müllerian hormone protein in the cytoplasm. According to RT-PCR results, these cells expressed Sertoli cell markers (FSHR, KRT18, and GATA6) and endogenous transcription factors genes (KLF4 and c-MYC). A total of 240 colonies (0.3% efficiency) were detected by day 7 after viral transduction of 72500 cells. The Sertoli iPSC-like colonies contained small cells with a high nucleus-to-cytoplasm ratio. These colonies tested positive for alkaline phosphatase staining, expressed endogenous pluripotency genes, and had a normal karyotype. All these cell lines could form in vitro three-dimensional aggregates that represented three germ layers of embryonic-like cells. A total of two cell lines used for in vivo differentiation produced high-efficiency teratoma. In conclusion, Sertoli cells can efficiently serve as a novel cell source for iPSC reprogramming.

Rabbit induced pluripotent stem cells retain capability of in vitro cardiac differentiation.

Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos.

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 µM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 µM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 µM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 µM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4-8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.

Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D.

Pig induced pluripotent stem cell (piPSC) line was generated from embryonic fibroblast cells using retroviral transduction approaches carrying human transcriptional factors: OCT4, SOX2, KLF4, c-MYC and LIN28. The generated piPSC line, VSMUi001-D, was positive for alkaline phosphatase activity and expressed the pluripotency associated transcription factors including OCT4, SOX2, NANOG and surface markers SSEA-1, all iPSC hallmarks of authenticity. Furthermore, VSMUi001-D exhibited a normal karyotype and formed embryoid bodies in vitro and teratomas in vivo. Upon cardiac differentiation, VSMUi001-D displayed spontaneous beating and expressed cardiomyocyte markers, like cardiac Troponin T.

Establishment of a rabbit induced pluripotent stem cell (RbiPSC) line using lentiviral delivery of human pluripotency factors.

Rabbit Embryonic Fibroblast (RbEF) cells (from Hycole hybrid rabbit foetus) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the newly generated RbiPSC was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the spontaneous differentiation potential of the iPSC was also tested in vivo by teratoma assay. The iPSC line showed normal karyotype. The advantages of using RbiPSC are the easy access to primary material and the possibility to study the efficacy and safety of the iPSC-based therapies on a non-rodent animal model.

Epigenetic modulation on cat-cow interspecies somatic cell nuclear transfer embryos by treatment with trichostatin A.

This study aimed to determine the acetylation at lysine 9/18/23 of histone H3 (H3K9ac/H3K18ac/H3K23ac; H3K9/18/23 ac) and the di-methylation at lysine 9 of histone H3 (H3K9me2) during early embryogenesis among trichostatin A (TSA)-treated interspecies somatic cell nuclear transfer (iSCNT) cat-cow (TSA-iSCNT) embryos, TSA-untreated iSCNT cat-cow control (control) embryos and bovine in vitro fertilization (IVF) embryos, because TSA-iSCNT embryos can develop to blastocysts. Compared to the control embryos, higher expressions of H3K9/18/23 ac were observed in TSA-iSCNT embryos and IVF embryos at most following stages (2 h post-fusion / post-insemination (PF/PI) to eight-cell stage). At 6 h PF/PI the expression of H3K9/23 ac in TSA-iSCNT embryos and IVF embryos were lower than those in control embryos, and the expression of H3K18ac was no difference among the three groups. The expression of H3K9/23 ac increased in TSA-iSCNT embryos and IVF embryos at pronuclear (PN) stages. The expression of H3K9me2 in TSA-iSCNT embryos resembled that of IVF embryos at 2 h PF/PI to PN stages, and these expression levels were greater than those of control embryos. These results suggest that treatment of iSCNT embryos with TSA modifies the patterns of histone modification at certain lysine residues in a manner that is comparable with that seen in IVF during early embryogenesis.

Influence of cooling and thawing conditions and cryoprotectant concentration on frozen-thawed survival of white-naped crane (Antigone vipio) spermatozoa.

To assist in genetic resource management and recovery efforts of the white-naped crane (Antigone vipio), we conducted two experiments to evaluate the effect of cooling condition, thawing rate, and cryoprotectant concentration on sperm survival post-thaw. Semen was collected from four mature males during breeding season (March and April) and evaluated for volume, sperm concentration, motility, and membrane integrity. In Experiment 1, ejaculates (n = 8) were diluted with Beltsville Poultry Semen Extender (BPSE) containing 10% dimethylsulfoxide (Me2SO) and frozen using either one (average cooling rate = 2.5 °C/min) or two step (average cooling rate = 7 and 9 °C/min, respectively) cooling method. The frozen samples were thawed using one of two thawing rates: 37 °C 30 s vs. 4 °C 1 min. In Experiment 2, samples were diluted with crane semen extender containing either 6% or 10% Me2SO, frozen using two-step method and then thawed at 37 °C for 30 s. Both cooling condition (two-step > one-step) and thawing rate (37 °C 30 s > 4 °C 1 min) impacted sperm motility, progression and kinetic characteristics (P < 0.05), but did not (P > 0.05) affect plasma membrane or acrosomal integrity. Concentration of Me2SO did not impact frozen-thaw survival. We conclude that white-naped crane sperm cryopreserved using a combination of two-step cooling and thawing at 37 °C 30 s was superior to other cooling and thawing combinations regarding to sustaining sperm motility with good motility kinetics. Findings represent the first steps towards the development of effective cryopreservation protocols and establishment of a genome resource bank for this threatened species.

Reproductive seasonality and sperm cryopreservation in the male tufted deer (Elaphodus cephalophus).

The tufted deer is a small deer, listed as near threatened on the International Union for Conservation of Nature Red List, and there is no information on the fundamental reproductive biology of this species. In this study, we report for the first time, characterization of male reproductive traits and cryopreservation of semen in this species. Males were subjected to electroejaculation during each season (autumn, winter, spring, and summer), and ejaculates were assessed for motility and quality traits. Fecal samples were collected 3 to 5 times weekly for 2 years and analyzed for androgen metabolites using enzyme immunoassay. Ejaculates with greater than 70% motility were cryopreserved using Beltsville extender (BF5F) or Triladyl. Straws were thawed and assessed subjectively as well as swim-up processed to isolate motile spermatozoa for computer-assisted sperm analysis and acrosome integrity at hourly interval. Tufted deer male reproductive and semen traits peaked in autumn. Mean fecal androgen concentrations were highest in the summer compared with baseline values during rest of the year. Sperm motility and acrosome integrity were lower immediately after thawing in both cryodiluents compared with raw ejaculates. Motility characteristics after swim-up were higher in BF5F compared with Triladyl. Four hours after thawing, both percent sperm motility and progression decreased further and were similar between BF5F and Triladyl. However, the proportion of spermatozoa with intact acrosomal membranes was higher in BF5F than Triladyl. Results indicate that tufted deer exhibit seasonal variations in reproductive traits and that BF5F better preserves sperm motility and acrosomal integrity after cryopreservation compared with Triladyl.

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.

Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus).

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2-4-cell embryos, 8-16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.

Spermatozoa isolated from cat testes retain their structural integrity as well as a developmental potential after refrigeration for up to 7 days.

The objective of this study was to compare the efficiency of preservation media for isolated feline testicular spermatozoa as well as the concentrations of bovine serum albumin (BSA) on: (1) the membrane (sperm membrane integrity (SMI)) and DNA integrity of spermatozoa; and (2) the developmental potential of spermatozoa after intracytoplasmic sperm injection (ICSI). Isolated cat spermatozoa were stored in HEPES-M199 medium (HM) or Dulbecco's phosphate-buffered saline (DPBS) at 4°C for up to 7 days. Results indicated that HM maintained a better SMI than DPBS throughout the storage periods (P > 0.05). When spermatozoa were stored in HM supplemented with BSA at different concentrations (4, 8 or 16 mg/ml), SMI obtained from HM containing 8 and 16 mg/ml BSA was higher than with 4 mg/ml BSA (P 0.05). In summary, cat spermatozoa immediately isolated from testicular tissue can be stored as a suspension in basic buffered medium at 4°C for up to 7 days. BSA supplementation into the medium improves membrane integrity of the spermatozoa during cold storage. Testicular spermatozoa stored in HM containing 16 mg/ml BSA retained full in vitro developmental potential after ICSI, similar to that of fresh controls even though DNA integrity had slightly declined.

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen.

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10(6) sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green- and CT-Red-labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10(6)) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).

Selective TGF-ß1/ALK inhibitor improves neuronal differentiation of mouse embryonic stem cells.

The transforming growth factor-ß1 (TGF-ß1), a polypeptide member of the TGF-ß superfamily, has myriad cellular functions, including cell fate differentiation. We hypothesized that suppression of TGF-ß1 signaling would improve the efficacy of neuronal differentiation during embryoid body (EB) development. In this study, mouse embryonic stem cells (ESCs) were allowed to differentiate into their neuronal lineage, both with, and without the TGF-ß1 inhibitor (A83-01). After 8 days of EB suspension culture, the samples were examined by morphological analysis, immunocytochemistry and immunohistochemistry with pluripotent (Oct4, Sox2) and neuronal specific markers (Pax6, NeuN). The alteration of gene expressions during EB development was determined by quantitative RT-PCR. Our results revealed that the TGF-ß1/ALK inhibitor potentially suppressed pluripotent gene (Oct4) during a rapidly up-regulation of neuronal associated genes including Sox1 and MAP2. Strikingly, during EB development, the expression of GFAP, the astrocyte specific gene, remarkably decreased compared to the non-treated control. This strategy demonstrated the beneficial function of TGF-ß1/ALK inhibitor that rapidly and uniformly drives cell fate alteration from pluripotent state toward neuronal lineages.

Insulin-like growth factor-1 (IGF-1) enhances developmental competence of cat embryos cultured singly by modulating the expression of its receptor (IGF-1R) and reducing developmental block.

OBJECTIVE: The aim of this study is to determine the effects of insulin-like growth factor-1 (IGF-1) and the mRNA expression of IGF-1 receptor (IGF-1R) during the in vitro development of cat embryos cultured in groups versus singly. METHODS: Cumulus-oocyte complexes (COCs) were matured and fertilized in vitro with frozen-thawed semen. Cleaved embryos (48h post-fertilization) were randomly assigned to one of the following treatments: 1) group embryo culture without IGF-1 (10 embryos per 50µl droplet), 2) single-embryo culture without IGF-1, and 3) to 6) single-embryo culture (50µl droplet per embryo) supplemented with different concentrations of IGF-1 (5, 25, 50 and 100ng/ml, respectively). During in vitro culture, the embryos were analyzed for development to the morula, blastocyst and hatching blastocyst stage. Relative mRNA expression of IGF-1R was also examined by qPCR at the morula and blastocyst stages. In addition, the mRNA expression of IGF-1R in morula-stage embryos treated with IGF-1 was determined. The influence of IGF-1 to preimplantation embryo development was then explored by co-incubation with 0.5µM IGF-1R inhibitor (Picropodophyllin; PPP). RESULTS: Group embryo culture led to a significantly higher blastocyst development rate compared with single-embryo culture (P<0.05). The poor development of singly cultured embryos coincided with the significantly lower IGF-1R expression in morulae than in group-cultured morulae. IGF-1 (25 or 50ng/ml) supplementation significantly improved the blastocyst formation rate of single embryos to a level similar to group culture by promoting the morula-to-blastocyst transition. IGF-1 supplementation (25 or 50ng/ml) of singly cultured embryos upregulated the expression of IGF-1R mRNA in morula-stage embryos to the same level as that observed in group-cultured embryos (without IGF-1). The beneficial effects of IGF-1 on singly cultured embryo were (P<0.05) suppressed by PPP even in the group culture embryo without growth factor supplementation. CONCLUSION: IGF-1 supplementation improves the developmental competence of feline embryos cultured individually and also increases IGF-1R gene expression to levels similar to group-cultured embryos.