Do the blood anti-Müllerian hormone concentrations in young females correlate with the ovarian follicular population later in life in water buffalo?

In this longitudinal study, the anti-Müllerian hormone (AMH) levels in blood were determined in 32 Murrah buffalo females at 8, 10, 12, 16 and 19 months of age when females were synchronized and the antral follicular population (AFP) was estimated. Correlations of AFP to the AMH level at 19 months of age and retrospectively to younger ages were investigated. Then females were split into high and low AFP, and their AMH levels were compared for all ages and tested as predictors of AFP categories. The highest AMH level (p < .05) was detected at 8 months, reducing but not differing (p > .05) at 10, 12 and 16 months then reducing again (p < .05) at 19 months of age. The mean AFP was 17.6 ± 6.3 follicles, and it was positively correlated with AMH in all ages tested. High AFP females had approximately two times more antral follicles than low AFP (p < .05) and their AMH levels were higher (p < .01) than in low AFP ones in all ages. Only at 8 months, AMH levels can be used to precociously detect high AFP heifers (a cut-off point of 464.7 pg/mL; p < .05), while low AFP heifers could be detected by AMH measurements at 8, 10, 12 and 16 months of age (p < .05). We conclude that AMH of buffalo calves correlates with AFP of heifers later in life and depending on the age, its levels could be used to identify future females with low or high AFP.

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and the development of therapies, as they can proliferate indefinitely under defined conditions and differentiate into any cell type in the body. Large-scale expansion of cells is limited in adherent culture, making it difficult to obtain adequate cell numbers for research. It has been previously shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem cells. Pigs are important preclinical models for stem cell research. Therefore, this study investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using an established porcine iPSC (piPSC) line as well as a new cell line derived and characterized in the current study, we report that piPSCs can grow in SSB while maintaining characteristics of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static culture. This culture method provides a suitable platform for scale-up of cell culture to provide adequate cell numbers for future research applications involving piPSCs.

Highly dynamic temporal changes of TSPY gene copy number in aging bulls.

The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.

Effect of cortisol on bovine oocyte maturation and embryo development in vitro.

Glucocorticoids (GCs) are important mediators of key cellular events. Herein, we investigated the effect of adding cortisol to the IVM medium on the acquisition of developmental competency in bovine oocytes. Cortisol (0.01, 0.1, or 1 µg/mL) had no effect on cleavage rates or cell numbers of resulting blastocysts; however, supplementation with 0.1 µg/mL during IVM increased blastocyst rates of in vitro-fertilized bovine oocytes as compared to untreated controls (41 ± 10% vs. 21 ± 1.2%, P < 0.05, respectively). This concentration was chosen to assess changes in the relative expression of potential GC target genes. Oocytes matured in the presence of cortisol and their corresponding cumulus cells did not show changes in expression for genes analyzed as compared to untreated controls. Notably, blastocysts from oocytes matured in cortisol-supplemented medium expressed higher relative levels of glucose transporter 1 (GLUT1), fatty acid synthase (FASN), and heat shock protein 70 (HSP70). This study supports a role for cortisol in the acquisition of bovine oocyte competence. This is evidenced by increased blastocyst development rates and presumably related to elevated embryonic transcripts with roles in glucose and lipid metabolism, as well as the cellular response to stress.

The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant.

The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle.

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality.

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.