Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.

When Chu-Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 51C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triplestain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology.

Fecal 17beta-estradiol and progestogens excretion was monitored in adult, female cheetahs (Acinonyx jubatus; n = 2), ZGG-12301 (born 3 April 1993), gonadotrophin treated and ZGT-3301, (born 19 August 1993), nontreated, for 120 days using commercially available plate enzyme immunoassay kits prepared for human serum or plasma. There were significant differences (P < 0.001) between baseline and peak concentrations of both hormone measures. Female ZGG-12301, which conceived, but this pregnancy resulted in an unobserved spontaneous abortion, showed no significant difference (P > 0.05) between baseline and gestation 17beta-estradiol values; fecal 17beta-estradiol excretion during pregnancy was statistically different (P < 0.001) from excretion during the nonpregnancy period. Baseline progestogen concentrations were different from pregnancy (P < 0.001) and postovulatory (P < 0.01) concentrations, and progestogen concentrations during pregnancy period were different (P < 0.001) from postovulatory concentrations. In the nontreated cheetah (ZGT-3301), basal and increased progestogen concentrations were statistically different (P < 0.01). On the basis of 17beta-estradiol excretory patterns, duration of the estrous cycle (x +/- SEM) was 13.2 +/- 2.2 days. These results suggest that the enzyme-linked immunosorbent methods reported in this study were capable of quantifying reproductive hormones in fecal extracts of cheetahs and could be a practical alternative to other enzyme-linked immunosorbent assays which require more complex procedures.

Barbary sheep (Ammotragus lervia) are considered vulnerable species by the World Conservation Union (IUCN). The purpose of this study was to describe the basic characteristics of fresh semen, test the efficacy of commercial extender Triladyl, and collect necessary data that may help to create a frozen semen bank for the Barbary sheep in Spain. A total of 21 ejaculates were collected by rectal-probe electroejaculation from one dominant (D) and three minor (M) adult males housed in the Madrid Zoo. After ejaculation, semen volume, concentration, and mass motility were assessed. Remaining raw semen was diluted at 37°C with TRIS-based extender Triladyl (Minitüb, Tiefenbach, Germany) and 20% egg yolk to a final concentration of 200 × 106 sperm per mL. Diluted samples were kept at 5°C for 4 h and then loaded into 0.25-mL French straws, frozen at 5 cm above liquid nitrogen (LN2) for 10 min and then plunged into LN2. Samples were thawed in a water bath at 37°C for 30 s. Post-thaw semen survival was evaluated by sperm motility (%M), quality of movement (Q), normal acrosome status (%NAS), normal sperm morphology (%NOR), membrane integrity (hypo-osmotic test; %HOST), and sperm viability (eosin-nigrosin vital staining; %V), and were compared with the same parameters in the fresh semen. Data between D and M males were analyzed by one way ANOVA. Mean volume of ejaculates, total sperm concentration and mass motility of raw semen were respectively; 5.2 ± 1.56 mL, 2800.0 ± 1290.5 × 106 and 3.4 ± 0.4 for the D male, and 3.5 ± 3.2 mL, 251.2 ± 103.9 × 106, and 1.88 ± 1.4 for M males (P < 0.05). Remaining semen parameters evaluated in raw semen showed no differences between D and M males. However, post-thaw semen quality was significantly (P < 0.05) reduced in all analyzed parameters except %NAS and %NOR in M males groups as compared to the D male (Table 1). It can be concluded that Barbary sheep raw semen collected by electroejaculation is of sufficient quality to be used in an artificial insemination program and can be successfully frozen in commercially available Triladyl extender. However, the post-thaw viability of semen may considerably depend on the male reproductive status in the flock. Table 1. Characteristics of fresh and cryopreserved Barbary sheep semen This work was supported by CAM 07B/007/1999 (Analysis of ejaculate traits and development of methods of semen preservation in wild ungulates from the Madrid Zoo).

The objective of the present study was to describe the basic characteristics of brown bear spermatozoa collected by electroejaculation and from the cauda epididymis after necropsy. Semen was collected during the mating season of 2004 and 2005 from five mature males, aged from 3.5 to 22 years. Animals were anesthetized with 7 to 10 mg/kg body weight of Zoletil® (tiletamine + zolazepam; Virbac, Carros, France). Bears were placed on a special stretcher in ventral recumbency. A rectal probe with three longitudinal electrodes and a diameter of 2 cm (Electrojac IV; Minitübe, Tiefenbach, Germany) was placed into the rectum. Two series of stimuli were administered with a 2–3 min rest between series. Each stimulus lasted 2 s and was administered at 2 s intervals increasing intensity of stimulation from 0 to 15V. Of 23 electroejaculation attempts, 17 ejaculates were collected and two were eliminated because of poor semen quality. Epididymal spermatozoa were collected from three animals after euthanasia by flushing cauda epididymis with the freezing medium. Two animals showed unilateral testicular hypoplasia and only one testicle was processed from each of them. Volumes were recorded and concentration was evaluated in the laboratory. Semen quality of epididymal and ejaculated samples was assessed by: progressive sperm motility (%IM); quality of movement on a scale of 0 to 5 (Q); normal acrosome status (%NAS) and normal sperm morphology (%NOR), assessed by phase contrast microscopy of glutaraldehyde fixed samples; membrane response to hypo-osmotic test (%HOST); and sperm viability estimated using eosin-nigrosin vital staining (%V). Results were evaluated by Student t-test. Mean volume of ejaculates was 0.79 ± 0.73 mL. The average concentration of samples collected by electroejaculation was 519 ± 278 × 106 sperm/mL. The average number of spermatozoa collected per ejaculate was lower than the average number of spermatozoa collected from the epididymis, 476 ± 352 × 106 vs. 640 ± 164 × 106, respectively (P > 0.05). Sperm quality was not different between epididymal and electroejaculated samples (Table 1), except that the %NORwas higher in electroejaculated samples than in epididymal samples (62 ± 16 vs. 3 ± 15; P < 0.01). Ahigh percentage of spermatozoa with abaxial midpiece attachment (78.6 ± 10.4%) was observed in all samples. All electroejaculates contained leukocytes and 53% of the ejaculates had agglutinated spermatozoa. All 15 ejaculate and all epididymal samples were frozen in TES-Tris extender containing 4% glycerol using a standard protocol for bull semen. Our results indicate that electroejaculated and epididymal brown bear spermatozoa may be of sufficient quality for use in AI programs to increase gene flow between isolated captive populations. Whether cryopreserved samples can be used for artificial breeding remains to be determined. Table 1. Quality parameters of spermatozoa from ejaculated and epididymal samples (mean ± SD) This work was supported by Zoological Society of San Diego.