Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis).

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n=6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 x 10(6)/mL) and motility (79%). Post-thaw sperm characteristics were higher (P<0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.

Compositional analysis for identification of arson accelerants by electron ionization Fourier transform ion cyclotron resonance high-resolution mass spectrometry.

Elemental compositions of each of 100 to 500 different constituents (i.e., every peak in a mass-to-charge ratio range, 50 < m/z < 300) of lighter fluid, kerosene, turpatine, gasoline, diesel fuel, and two brands of mineral spirits (and their weathered analogs) make possible direct identification of each accelerant in a experimental fire, based on electron ionization 6.0 Tesla Fourier transform ion cyclotron resonance (EI FT-ICR) ultrahigh resolution mass spectrometry. Septum injection of as little as 500 nL of accelerant into an all-glass heated inlet system yields definitive elemental compositions (molecular formulas) based on accurate (< +/-1 ppm average error) mass measurement alone. Extraction and EI FT-ICR mass analysis of fire debris from a controlled burn of a couch with simple (lighter fluid) and complex (turpatine) ignitable liquid yielded dozens of elemental compositions serving as a unique "fingerprint" for each petroleum product, despite the presence of up to 249 additional extracted matrix and pyrolysis components. Forty-five of 56 lighter fluid constituents and 126 of 133 turpatine constituents (not counting 13C-containing species) were identified in the debris from a fire staged for each respective accelerant.

Stable isotope incorporation triples the upper mass limit for determination of elemental composition by accurate mass measurement.

By comparing electrospray ionization Fourier-transform ion cyclotron resonance (FT-ICR) mass spectra and collision-induced dissociation (CID) FT-ICR mass spectra of a phospholipid (851 Da) extracted from natural abundance and 99% 13C bacterial growth media, we are able to reduce its number of possible elemental compositions (based on +/-10 ppm externally calibrated mass accuracy and biologically relevant compositional constraints) from 394 to 1. The basic idea is simply that the mass of a molecule containing N carbon atoms increases by N Da when 12C is replaced by 13C. Once the number of carbons is known, the number of possible combinations of other atoms in the molecule is greatly reduced. We demonstrate the method for a stored-waveform inverse Fourier transform-isolated phospholipid from an extract of membrane lipids from Rhodococcus rhodochrous hydrocarbon-degrading bacteria grown on either natural abundance or 99% 13C-enriched mixtures of n-hexadecane and n-octadecane. We project that this method raises the upper mass limit for unique determination of elemental composition from accurate mass measurement by a factor of at least 3, thereby extending "chemical formula" determination to identification and sequencing of larger synthetic and bio-polymers: phospholipids, oligopeptides of more than three to four amino acids, DNA or RNA of more than two nucleotides, oligosaccharides of more than three sugars, etc. The method can also be extended to determination of the number of other atoms for which heavy isotopes are available (e.g., 15N, 34S, 18O, etc.).

Melting corneal ulcer management in a greater one-horned rhinoceros (Rhinoceros unicornis).

Acute unilateral keratomalacia, probably secondary to trauma, occurred in a greater one-horned rhinoceros (Rhinoceros unicornis) transferred between zoologic facilities. Following 2 days of medical treatment, a 360 degrees conjunctival surgical graft was performed. Staphylococcus and yeast were isolated from a perioperative culture of the affected eye and were treated with antimicrobials. There was rapid healing and minimal midcorneal scar formation with peripheral corneal clarity.

Comparing ovulation synchronization protocols for artificial insemination in the scimitar-horned oryx (Oryx dammah).

Ovarian response and pregnancy success in scimitar-horned oryx (n=28) were compared, following treatment with two synchronization protocols and fixed-time artificial insemination (AI) with frozen-thawed semen. Each oryx received two injections of 500 microg of prostaglandin-F(2alpha) analogue (PGF(2alpha)-only) 11 days apart, and half received PGF(2alpha) in combination with an intravaginal progesterone-releasing device (CIDR11+PGF(2alpha)). Semen was collected by electroejaculation from anaesthetised adult oryx and cryopreserved. Anaesthetised females were transcervically inseminated 56.0+/-1.1 h (+/-S.E.M.) after PGF(2alpha) injection and/or device withdrawal using 28.0+/-1.5x10(6) motile thawed sperm. Ovarian endocrine response was monitored in 20 females by analysing faecal oestrogen and progesterone metabolites. Periovulatory oestrogen peaks were detected in 19/20 (95%) females after synchronization. There were no between-treatment differences in oestrogen concentrations or peak characteristics (P0.05). Luteal development after synchronization was delayed in half the progesterone treated (CIDR11+PGF(2alpha)) females, and faecal progestin excretion profiles indicated that the ovulatory follicle associated with synchronization either failed to ovulate or to fully lutenise. Pregnancy was diagnosed by ultrasonography and/or rectal palpation and was monitored by faecal progestin excretion. More (P=0. 013) pregnancies resulted from the PGF(2alpha)-only treatment (37.5%, 5/14) than from the CIDR11+PGF(2alpha) treatment (0/14), and four healthy scimitar-horned oryx calves were born, three after gestation intervals of 247 days and one after 249 days.

Enhancing zona penetration by spermatozoa from a teratospermic species, the cheetah (Acinonyx jubatus).

Cheetahs (Acinonyx jubatus) produce poor quality ejaculates that can limit the efficiency of standard assisted reproduction including artificial insemination (AI) and in vitro fertilization (IVF). The purpose of this study was to: (1) further study sperm-oocyte interaction in this teratospermic species by examining the ability of malformed sperm to interact with various oocyte barriers; and (2) assess the potential of zona piercing for assisting IVF in a teratospermic felid. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Semen was collected by electroejaculation from six male cheetahs and ejaculates were processed for IVF. Sperm aliquots from each ejaculate were assessed for a sperm motility index (SMI) over time. Zona-intact (ZnIn-SS) oocytes (n = 78) and ZnPd-SS oocytes (n = 74) were coincubated with spermatozoa in vitro for 6 h. The proportion of morphologically abnormal spermatozoa per ejaculate was high for all males (range 81.5% to 95.9%). SMI values at 0 and 6 h were variable, ranging from 50 to 75 and 0 to 40, respectively. Spermatozoa from all ejaculates bound to and penetrated the outer zona pellucida of ZnIn-SS and ZnPd-SS oocytes similarly (P > 0.05). The proportion of oocytes containing spermatozoa within the inner zona layer and the average number of spermatozoa per oocyte in this region were greater (P < 0.05) for the ZnPd-SS than ZnIn-SS oocytes (39.2% and 1.0 versus 12.8% and 0.2, respectively). Although zona piercing enhanced sperm penetration, there was no increase (P > 0.05) in pleiomorphic spermatozoa penetrating the inner zona pellucida or PVS.(ABSTRACT TRUNCATED AT 250 WORDS)

The cotton-top tamarin revisited: Mhc class I polymorphism of wild tamarins, and polymorphism and allelic diversity of the class II DQA1, DQB1, and DRB loci.

Cotton-top tamarins (Saguinus oedipus) in captivity are unusual in that they exhibit low levels of polymorphism and allelic diversity at the major histocompatibility complex (Mhc) class I loci. Since the polymorphism has previously only been examined in captive tamarins, we analyzed the Mhc class I alleles of a population of wild tamarins. These wild tamarins, like their captive counterparts, exhibited limited class I polymorphism. We also assessed the levels of polymorphism and allelic diversity at the Mhc class II DQA1, DQB1, DQB2, and the DRB loci in captive populations of cotton-top tamarins. In contrast to the extensive polymorphism in Old World monkeys, only two alleles were detected at each of DQA1 and DQB1. Also, the DQB2 locus was monomorphic and conserved between New and Old World monkeys. Sequences derived from four putative DRB loci were obtained, and extensive polymorphism was found at all four loci. Phylogenetic analysis did not indicate that any of the tamarin DRB loci, with the possible exception of Saoe-DRB3, were orthologous to the human DRB loci. At three of the DRB loci (Saoe-DRB11, Saoe-DRB*W12, Saoe-DRB*W22), the number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites, indicative of positive selection. We found no support for a restriction on the polymorphism at the cotton-top tamarin class II loci. However, the allelic diversity at some of the Saoe-DRB loci is more limited than for the HLA-DRB1, consistent with a restriction imposed by the bone marrow-chimerical lifestyle.

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.