Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope.

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams.

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.

A review of the promises and pitfalls of oocyte and embryo metabolomics.

Embryo viability assessment is one of the most important and challenging tasks in IVF. Evaluation of embryo quality is critical when selecting the best embryo(s) to transfer or cryopreserve. Until recently, the only instrument used for embryo evaluation was the inverted light microscope, which provided information based on morphological characteristics. Developmental and morphological information gained from microscopic assessment have been positively associated with IVF outcomes, including pregnancy and implantation rates. However, based on general statistics, it is clear that IVF currently still results in relatively low pregnancy rates, while simultaneously being associated with relatively high multiple implantation rates. Only with novel embryo assessment and selection procedures would it be possible to improve these outcomes. Accordingly, it has been proposed that it is possible to test the culture environment of a developing embryo to gain valuable information regarding its viability. Different approaches have been used. These include the measurement of oxygen consumption by the embryo and testing of the soluble HLA-G in the environment, as it was proposed that secretion of HLA-G is associated with higher implantation rates. Amino acid turnover, which appears to be correlated to blastocyst development, can be measured as an indication of embryo viability. Other approaches, such as time-lapse video observation or cumulus cell gene expression analysis, may be used in the future to gain a broader understanding of embryo viability. Proteomics and metabolomics are also useful tools for assessment of embryo developmental potential. Results from recent studies on predicting embryo viability by analyzing the metabolome of different stage embryos are promising, as increases in pregnancy and implantation rates were obtained using the metabolomic profile for embryo selection. Several novel approaches are currently being developed to aid in viability assessment. These need to be evaluated in prospective clinical trials, while considering their practicality in the clinical laboratory.