Germany/Australia index of sperm sex sortability in elephants and rhinoceros.

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.

New aspects of boar semen freezing strategies.

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

Production of piglets with sexed semen employing a non-surgical insemination technique.

The aim of the present study was to ascertain whether multiparous sows could successfully be inseminated with sexed semen non-surgically. Spermatozoa were stained with Hoechst 33342 and separated flowcytometrically in X- and Y-chromosome bearing sperm populations employing the Beltsville Sperm Sexing Technology (BSST). After weaning, estrus was induced in sows with PMSG and hCG. Animals were inseminated once per estrus non-surgically with a specially designed catheter into the tip of the uterine horn, employing 50x10(6) of either sexed or non-sexed spermatozoa diluted in 2 ml Androhep. Pregnant sows were allowed to go to term. Mean pregnancy rate from inseminations with unsexed spermatozoa was 54.5% whereas inseminations with sexed spermatozoa resulted in 33.3% pregnant sows. All but one piglet born after insemination with sexed semen were of the predicted sex. The sex of those piglets born after inseminations with non-sexed spermatozoa was 61.1% for male and 38.9% for female sex. It is concluded that non-surgically inseminations with flowcytometrically sexed spermatozoa can be conducted successfully.

Capillary anastomosis in vena caval thrombosis.

A 29-year-old woman presented with recurrent diffuse histiocytic lymphoma. Bilateral brachial venous catheters were placed for chemotherapy, which resulted in thrombotic occlusion of the superior vena cava. A lung scan with Tc-99m MAA (flow study and static images) demonstrated extensive collateral vessels in the upper thorax and trapping of particles in the right upper abdomen, anteriorly. A simultaneous liver scan with Tc-99m sulfur colloid (flow and delayed images) illustrated a normal liver with no other abnormal sites of tracer deposition. The accumulation of a lung imaging agent in the anterior abdominal wall was secondary to capillary anastomosis between the superior and inferior epigastric veins.

Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

Solid-cystic tumour of the pancreas. An endocrine neoplasm?

Immunohistochemical studies and DNA flow-cytometric investigations were performed in a case of solid-cystic tumour of the pancreas in a 35-year-old woman. All tumour cells were immunoreactive for the neuroendocrine cell markers chromogranin A and neuron-specific gamma-enolase. Moreover, about 10% of tumour cells were immunoreactive for insulin, while hypoglycaemia was absent. Few tumour cells (less than 1%) were immunoreactive for somatostatin, and no cells were found to be immunoreactive for pancreatic polypeptide or glucagon. No immunoreactivity was present for duct cell marker carcino-embryonic antigen and only individual cells were reactive for alpha 1-antitrypsin. Nuclear DNA content of the tumour cells was diploid and the proliferative activity was low. In confirmation of some reports on neuroendocrine markers in solid-cystic tumour of the pancreas, our findings support the theory that the lesion is a hormonally inactive neuroendocrine pancreatic tumour.

Proliferative activity of neuroendocrine tumours of the gastroenteropancreatic endocrine system: DNA flow cytometric and immunohistological investigations.

The proliferative activity of 16 tumour specimens from 13 patients with neuroendocrine tumours of the gastroenteropancreatic endocrine system was studied by DNA flow cytometry and immunohistology for the nuclear Ki67 proliferation antigen. Equivalent results were obtained with both methods, which showed the proliferative activity of gastroenteropancreatic neuroendocrine tumours to be heterogeneous. In four malignant small intestinal carcinoids and one extravisceral carcinoid localised in the retroperitoneum the percentage (index) of proliferating tumour cells as measured by DNA flow cytometry ranged from 2.9 to 36.2% corresponding to low, moderate, or high proliferative activity. In four malignant pancreatic endocrine tumours and their metastases indices ranged from 8.7 to 18.3%, corresponding to low, moderate, or high proliferative activity. In four benign pancreatic endocrine tumours indices ranged from 4.3 to 7.7%, all corresponding to low proliferative activity. This heterogeneity of proliferative activity may in part explain the heterogeneous results reported of chemotherapy treatment. As chemotherapy of tumours is largely affected by favourable cell cycling kinetics, individual diagnostic investigations of the proliferative activity of these neuroendocrine tumours may be of value for identifying patients suitable for this treatment.