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Recent success propagating captive beluga has resulted from combined efforts by North American zoos and aquariums to manage disparate collections as a single population. This success has provided a tremendous opportunity to increase our understanding of beluga reproductive biology. Blood samples were collected on a weekly to biweekly basis from 23 female and 12 male beluga, ranging in age from 2–15 years, for analysis of serum progesterone (P) and testosterone (T), respectively. Peri-parturient observational data, including food intake, duration and signs of labor, and nursing patterns were collected from 15 days prepartum to 30 days postpartum during 21 births. Total body lengths and weights were collected from 10 captive-born beluga. For female beluga, the mean (±SD) age, body length, and weight at first conceptions were 9.1±2.8 years, 318.0±9.1 cm, and 519±84 kg. Thirty-five luteal phases and 13 conceptions were detected from January–June, and 70% of luteal phases and 80% conceptions occurred from March–May. The mean luteal phase and total estrous cycle lengths were 30.0±6.5 days and 48.0±4.6 days, respectively. For male beluga, the mean age that males sired their first calf was 13.3±2.6 years. Compared to younger males ( 8 years of age, 5.0 ng/ml) were elevated significantly only during the interval from January–April. Highest T concentrations (6.2±4.9 ng/ml) were recorded from January–March, whereas nadir concentrations (1.1±1.0 ng/ml) were detected from August–September. The mean gestation length was 475.0±20.4 days (n=9). For parturition, the mean time from the first appearance of fluke or rostrum to delivery, delivery to placental passage, and delivery to nursing were 4.4±2.9 hr, 7.6±1.8 hr, and 43±45 hr, respectively. All cows had decreased food intake on the day of delivery, with 44% having zero intake. Peak 24-hr nursing activity occurred 3.9±2.7 days post-partum. Growth (i.e., body weight and length) as a function of age were well described by the Gompertz model (r2=0.91, 0.93). Based on the model, growth in body weight and length were significantly greater in males compared to females. Predicted birth weight (88.9 kg) was similar for both sexes, however, and male calves were predicted to be shorter (154.3 cm) than female calves (160.7 cm). The results provide the first descriptions of captive beluga reproductive physiology, including endocrinology, peri-parturient behavior, growth, and reproductive maturity. This knowledge is important for helping to maintain genetically diverse, self-sustaining populations of captive beluga whales. Zoo Biol 24:29–49, 2005. © 2005 Wiley-Liss, Inc.

Presumptive copper deficiency was diagnosed in hand-reared captive pronghorn (Antilocapra americana) at the Los Angeles Zoo. Clinical signs, which were manifested in growing fawns, included anemia, anorexia, diarrhea, progressive paresis/recumbency, and aortic rupture. The range of serum copper concentrations in fawns born during the 1989 season (0.08-0.67 ppm) was below levels considered normal for domestic sheep and goats (0.7-2.0 ppm) and below concentrations measured in adult pronghorn (0.4-1.43 ppm). Copper sulfate supplementation of the hand-rearing formula, which was initiated in 1989, resulted in a significant increase in mean (+/- SD) serum copper levels from 0.45 +/- 0.18 ppm before supplementation to 0.68 +/- 0.05 ppm after supplementation (P0.05). Fawns born in subsequent seasons (April 1990-August 1993) continued to be supplemented with copper in the hand-rearing formula. Mean serum copper concentration from these fawns (0.68 +/- 0.22 ppm) was similar to the mean values from supplemented 1989 fawns and adult pronghorn in this herd (0.85 +/- 0.34 ppm; P0.05). No clinical signs of copper deficiency were detected in any fawns after supplementation was started. Analyses of the herd's diet revealed marginal dietary copper levels. Suspected dietary deficiency was confirmed by marginal tissue and serum copper concentrations in some of the herd's adult animals. Dietary copper levels were corrected to prevent future cases of clinical copper deficiency.

Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g., Ryan and Higgins, 1993, J Cell Physiol 155:376–384; Mu et al., 1998, J Cell Physiol 174:90–98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G0) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth “activated” phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G0G1 transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-β1 (TGF-β1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-β1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-β1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-β1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-β1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-β1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G0. These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene. J. Cell. Physiol. 181:96–106, 1999. © 1999 Wiley-Liss, Inc.

Disruption of the cell-cycle regulation through over-expression or mutation of cyclins and cyclin-dependent kinases has been implicated in carcinogenesis. In order to determine whether keratoacanthoma (KA) is unique or a variant of squamous cell carcinoma (SCC) and whether expression of mitosis-related antigens are associated with KAs' tendency to regress, we compared the immunohistochemical expression of mitotic cyclins (cyclins A and B) and their cyclin-dependent kinase p34(cdc2) in 21 KAs, 8 regressing KAs, and 28 conventional squamous cell carcinomas. KAs showed both overlap and significant differences in expression of these mitosis-related antigens compared to SCCs. Basal and parabasal pattern of expression of cyclins A and B significantly predominated in KAs in contrast to SCCs which exhibited diffuse pattern (cyclin A 86%/cyclin B 64% vs. 25%/36%, p0.01). However, no differences in the highest mean level of expression in 'hot spot' loci of cyclins A and B were identified comparing KAs to SCCs (19%/12% vs. 25%/13%, p0.05). For the cyclin-dependent kinase p34(cdc2), no differences in pattern, distribution or mean levels of expression were found. For cyclins A and B, regressing KA showed significantly more regional tumor labeling (88%/88% vs. 57%/33%, p = 0.03) and a lower mean level of immunoreactivity (5%/4% vs. 19%/12%, p = 0.001) compared to mature KAs. These findings indicate a role for mitotic cyclins in the evolution of both SCC and KA. The overlapping patterns of expression for these mitosis-related antigens suggest that KAs represent a variant of SCC that exhibit an overwhelming but not absolute tendency to involute.

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10 000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.