Intraocular pressure (IOP) measurement is a common procedure during eye examinations in birds. Differences in the IOP between avian species have been reported, which suggests the need to establish species-specific reference ranges. To determine IOP values of captive black-footed penguins (Spheniscus demersus), we obtained IOP readings with the use of a rebound tonometer by using two established calibration settings (dog and horse). No difference was seen in the IOP between the left and right eye when the horse setting was used; however, a difference was present when using the dog setting. No significant difference between the IOP of male and female penguins was seen in both eyes when the dog or horse setting was used. Rebound tonometry appears to be a safe and repeatable method to obtain IOP values in black-footed penguins.

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.

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.

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The dominance hierarchy of a group of adult female elands (n ¼ 10) kept in captivity was followed for 34 months. Outcomes of dominance relationships at the beginning and end of the study were compared. A clear dominance hierarchy existed in the herd. The dominance pattern was complex, but triads were predominantly (95%) transitive or linear. Reversal of dominance occurred in 12 dyads (27%), of which eight (67%) involved a single female. Two females shared the most dominant rank at the beginning of the study. One of these two females and another female later assumed the highest dominance rank on different occasions. A single female remained most subordinate throughout the study period. The correlation between body weight and dominance rank was not significant (r ¼ 0.46; P ¼ 0.21). Similarly, dominance rank was not associated with the taming potential of the females. However, the median dominance value increased in females with good taming potential, while it decreased in those with poor taming potential. In conclusion, captive eland antelope have a dynamic and complex dominance hierarchy that is predominantly linear.

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mM) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

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.

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