Social and genetic connectivity despite ecological variation in a killer whale network.

Philopatric kin-based societies encourage a narrow breadth of conservative behaviours owing to individuals primarily learning from close kin, promoting behavioural homogeneity. However, weaker social ties beyond kin, and across a behaviourally diverse social landscape, could be sufficient to induce variation and a greater ecological niche breadth. We investigated a network of 457 photo-identified killer whales from Norway (548 encounters in 2008-2021) with diet data available (46 mixed-diet individuals feeding on both fish and mammals, and 411 exclusive fish-eaters) to quantify patterns of association within and between diet groups, and to identify underlying correlates. We genotyped a subset of 106 whales to assess patterns of genetic differentiation. Our results suggested kinship as main driver of social bonds within and among cohesive social units, while diet was most likely a consequence reflective of cultural diffusion, rather than a driver. Flexible associations within and between ecologically diverse social units led to a highly connected network, reducing social and genetic differentiation between diet groups. Our study points to a role of social connectivity, in combination with individual behavioural variation, in influencing population ecology in killer whales.

Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Auditory masking of tonal and conspecific signals by continuous active sonar, amplitude modulated noise, and Gaussian noise in killer whales (Orcinus orca).

Continuous active sonar is thought to mitigate severe acoustic impacts due to its lower sound pressure level compared to pulsed active sonar typically used by world navies. However, due to its almost continuous duty cycle, continuous active sonar could have a higher potential for auditory masking. Here, we evaluate the auditory masking potential of several noise types including a recording of continuous active sonar, amplitude modulated noise, and Gaussian noise, on signal detection in two killer whales. Signals were either a 1.5 kHz pure tone or a recording of a broadband burst-pulse killer whale call. For the 1.5 kHz tone, all noise types resulted in statistically significant masking, however, there was a release from masking of approximately 13 dB for the amplitude-modulated noise. When the killer whale call was the signal, the whales employed an off-frequency listening strategy where the whales were able to detect frequency components of the signal that did not directly overlap with the noise. However, this strategy was less useful for the continuous active sonar noise due to its broadband harmonic structure. Continuous active sonar has spectral features that considerably overlap with those of killer whale calls, making this type of noise an effective auditory masker.

Temporal integration of tone signals by a killer whale (Orcinus orca).

A psychophysical procedure was used to measure pure-tone detection thresholds for a killer whale (Orcinus orca) as a function of both signal frequency and signal duration. Frequencies ranged between 1 and 100 kHz and signal durations ranged from 50 µs to 2 s, depending on the frequency. Detection thresholds decreased with an increase in signal duration up to a critical duration, which represents the auditory integration time. Integration times ranged from 4 ms at 100 kHz and increased up to 241 ms at 1 kHz. The killer whale data are similar to other odontocete species that have participated in similar experiments. The results have implications for noise impact predictions for signals with durations less than the auditory integration time.

Killer whale innovation: teaching animals to use their creativity upon request.

Thinking flexibly is a skill that enables animals to adapt to changing environments, which enhances survival. Killer whales, Orcinus orca, as the ocean apex predator display a number of complex cognitive abilities, especially flexible thinking or creativity when it comes to foraging. In human care, smaller dolphins and other marine mammals have been trained to think creatively while under stimulus control. The results of these previous studies have demonstrated that bottlenose dolphins, Tursiops truncatus, can create original behaviors in response to an innovative cue. We trained and tested a total of nine killer whales from two different facilities on the innovate concept, using the same methodology. The killer whales ranged in age from 5 to 29 yrs with 4 females and 5 males. The results indicate that the killer whales demonstrated high fluency, originality, some elaboration, and flexibility in their behaviors. Individual variability was observed with younger animals demonstrating more variable behaviors as compared to the older animals. Males seemed to display less complex and lower energy behaviors as compared to females, but this impression may be driven by the age or size of the animal. These results support existing evidence that killer whales are dynamic in their thinking and behavior.

Auditory masking in killer whales (Orcinus orca): Critical ratios for tonal signals in Gaussian noise.

Masked detection thresholds were measured for two killer whales (Orcinus orca) using a psychoacoustic, adaptive-staircase procedure. Noise bands were 1-octave wide continuous Gaussian noise. Tonal signals extended between 500 Hz and 80 kHz. Resulting critical ratios increased with the signal frequency from 15 dB at 500 Hz up to 32 dB at 80 kHz. Critical ratios for killer whales were similar to those of other odontocetes despite considerable differences in size, hearing morphology, and hearing sensitivity between species.

Cardiorespiratory coupling in cetaceans; a physiological strategy to improve gas exchange?

In the current study we used transthoracic echocardiography to measure stroke volume (SV), heart rate (fH) and cardiac output (CO) in adult bottlenose dolphins (Tursiops truncatus), a male beluga whale calf [Delphinapterus leucas, body mass (Mb) range: 151-175 kg] and an adult female false killer whale (Pseudorca crassidens, estimated Mb: 500-550 kg) housed in managed care. We also recorded continuous electrocardiogram (ECG) in the beluga whale, bottlenose dolphin, false killer whale, killer whale (Orcinus orca) and pilot whale (Globicephala macrorhynchus) to evaluate cardiorespiratory coupling while breathing spontaneously under voluntary control. The results show that cetaceans have a strong respiratory sinus arrythmia (RSA), during which both fH and SV vary within the interbreath interval, making average values dependent on the breathing frequency (fR). The RSA-corrected fH was lower for all cetaceans compared with that of similarly sized terrestrial mammals breathing continuously. As compared with terrestrial mammals, the RSA-corrected SV and CO were either lower or the same for the dolphin and false killer whale, while both were elevated in the beluga whale. When plotting fR against fH for an inactive mammal, cetaceans had a greater cardiac response to changes in fR as compared with terrestrial mammals. We propose that these data indicate an important coupling between respiration and cardiac function that enhances gas exchange, and that this RSA is important to maximize gas exchange during surface intervals, similar to that reported in the elephant seal.

Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry.

There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehydroepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: progesterone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3ß,20α-dihydroprogesterone (3ß,20α-DHP; 40-80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3ß,20α-DHP were similar at mid-gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3ß,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (<10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3ß,20α-DHP is the major metabolite, increasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also increased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but progesterone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy.

Longitudinal profiles of relaxin and progestagens during pregnancy, pregnancy loss and false pregnancy in the killer whale (Orcinus orca).

The circulating pattern of immunoreactive relaxin and progestagens based on monthly and gestational stage (early, mid, late) profiles were determined during pregnancies that resulted in live calves (LIVE, n = 30), stillbirths (STILLB, n = 3), abortions (ABORT, n = 5) and presumptive false pregnancies (FALSE, n = 8), and during the follicular (n = 34) and luteal phase (n = 58). Monthly LIVE relaxin concentrations steadily increased during gestation, but values did not significantly exceed those of the luteal phase until 9 months prior to parturition, peaking during the final month at 2356 ng/ml. Relaxin surged (P < 0.05) during the final week of gestation (36,397 ng/ml), undergoing a 3 and 9-fold increase compared with concentrations in the preceding two weeks, respectively. Monthly relaxin production did not differ among each reproductive state with the exception of months-13-16 where concentrations were higher (P < 0.001) for STILLB than LIVE. Relaxin concentration was reduced (P < 0.0001) by 849% in placental versus maternal serum collected within 1 day of labor. Mid- and late-pregnancy progestagen concentrations were lower for FALSE (P < 0.001) compared with STILLB and LIVE. Late pregnancy progestagen concentrations were reduced for FALSE (P < 0.05) and ABORT (P < 0.02) compared with LIVE and STILLB. Monthly progestagen production in ABORT tended to be lower than LIVE across a range of gestational months (Months 2, 7, 8, 11) but this difference only became significant during months 14 and 15. Results indicate that relaxin is primarily produced by the CL during pregnancy, and that concentrations could not be used to differentiate from non-pregnant females until the final 6 months of gestation. In addition, as would be expected from a primarily CL product, relaxin cannot be used to detect abnormal pregnancies. Conversely, progestagens, which are produced by both the placenta and CL can be used to differentiate FALSE from normal pregnancy and may be useful indicators of fetal health in the killer whale.

SERUM COBALAMIN AND FOLATE CONCENTRATIONS AS INDICATORS OF GASTROINTESTINAL DISEASE IN KILLER WHALES ( ORCINUS ORCA).

Cobalamin and folate are water-soluble vitamins that are useful indicators of chronic gastrointestinal (GI) function in humans and some animal species. Serum cobalamin and folate concentrations in an ex situ population of killer whales ( Orcinus orca) were measured and factors that may affect their serum concentrations were identified. Serum samples ( n = 104) were analyzed from killer whales ( n = 10) both while clinically healthy and during periods of clinical GI disease as defined by clinical signs and fecal cytology. To characterize serum cobalamin and folate concentrations in clinically healthy animals, a mixed-model regression was used, with cobalamin and folate both significantly affected by weight (cobalamin: P < 0.0001, folate: P = 0.006) and season (cobalamin: P < 0.0001, folate: P < 0.0001). The marginal mean concentrations for cobalamin and folate across weight and season were 742 ± 53.6 ng/L and 30.2 ± 2.6 µg/L, respectively. The predicted 95% confidence intervals (CI) for these analytes were then compared with samples collected during periods of GI disease. Across individuals, 22% (2/9) of the folate and 80% (8/10) of the cobalamin samples from the animals with GI disease fell outside the 95% CI for the population. When comparing samples within an individual, a similar pattern presented, with 100% of cobalamin of the observed abnormal samples reduced compared to healthy animal concentration variability. The same was not true for folate. These results suggest that serum concentrations of cobalamin and folate may be useful minimally invasive markers to identify GI disease in killer whales, especially when values are compared within an individual.

Circulating anti-Müllerian hormone concentrations in relation to age and season in male and female beluga (Delphinapterus leucas).

The present study aimed to describe serum anti-Müllerian hormone (AMH) patterns of ex situ male and female beluga to examine the influence of age (divided into 5-year categories) or sexual maturation and reproductive season. In males aged 5-9 years, AMH concentrations were significantly (P<0.05) higher than those in all age categories exceeding 15 years and were not influenced by season (P=0.57). AMH concentrations in females peaked in the 5-9-year age category during the breeding season and decreased (P<0.05) after 9 years of age. Aged females displayed lower (P<0.05) AMH concentrations than immature and mature animals and immature females secreted higher concentrations than mature animals (P=0.03). For mature females, seasonal differences (P=0.02) in AMH concentrations were detected, with females in the breeding season displaying higher AMH concentrations than in the non-breeding season. This is the first time AMH has been characterised in a cetacean species and the first potential hormonal evidence of reproductive senescence in beluga. Further research is required to determine if this hormone can be used as a predictor of fertility for the species.

Characterization and longitudinal monitoring of serum androgens and glucocorticoids during normal pregnancy in the killer whale (Orcinus orca).

The secretory patterns of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA), cortisol (C), and corticosterone (Co) were characterized throughout 28 normal pregnancies until two-months post-partum in eleven killer whales. Effects of fetal sex, dam parity or age, and season were evaluated across either day post-conception (DPC), stage of pregnancy (PRE, EARLY, MID, LATE, POST) or indexed month post-conception (IMPC) using a mixed model linear regression with animal ID and pregnancy number as the random variables. Across DPC, DHEA, A4 and T concentrations were affected (P<0.05) by season, with highest concentrations during spring (DHEA, A4, & T) and summer (A4) as compared to the fall. A significant effect of parity on androgen production was observed only for DHEA, with multiparous females having higher (P=0.01) concentrations than nulliparous females. All three androgens significantly increased with each successive pregnancy stage and IMPC with peak concentrations occurring during IMPC 10 (DHEA), 13 (A4) and 14 (T), respectively. Cortisol was affected by season (P=0.03) with highest concentrations being detected during the months of fall, while Co was only affected by parity (P=0.003) with significant increases observed for primiparous females as compared to nulliparous females. Cortisol and Co concentrations peaked (P<0.05) during IMPC 17 (i.e., the month prior to parturition). The C to Co ratio during pregnancy was 7.4 to 1, indicating that cortisol is the major circulating glucocorticoid studied to date in pregnant killer whales. The significant increase in concentrations of maternal androgens throughout pregnancy, which were unrelated to fetal sex, indicates that they play an important role during killer whale fetal development.

Relaxin as a hormonal aid to evaluate pregnancy and pregnancy loss in bottlenose dolphins (Tursiops truncatus).

This study was conducted to critically evaluate weekly and monthly circulating concentrations of immunoreactive relaxin throughout pregnancies that resulted in live births, stillbirths, and abortions in aquarium-based bottlenose dolphins. A relaxin RIA was used to analyze serum collected during 74 pregnancies involving 41 dolphins and 8 estrous cycles as well as 8 non-pregnant dolphins. Pregnancies resulted in live births (n=60), stillbirths (n=7), or abortions (n=7). Relative to parturition (Month 0), monthly changes (P<0.0001) in relaxin was indicated by relatively low concentrations during early pregnancy (Months -12 to -9) which subsequently increased (P<0.05) during mid- (Months -8 to -5) to late (Months -4 to -1) pregnancy; relaxin was highest (P<0.05) at the time of parturition. Post-parturition (Month 1), concentrations decreased (P<0.05). During the first 4weeks post-ovulation, relaxin concentrations were not different between pregnant and non-pregnant dolphins (status-by-week interaction, P=0.59). Status-by-month interaction (P<0.0002) involving different pregnancy outcomes was due, impart, to an increase in relaxin during early pregnancy (P<0.05) that was comparable among dolphins with live births, stillbirths, and abortions except concentrations were lower (P<0.05; 52%) at mid-pregnancy in association with pregnancy loss. Thereafter, concentrations increased (P<0.05) during late pregnancy in dolphins with stillbirths but not in dolphins with abortions. In conclusion, this study provided new information on the pregnancy-specific nature of relaxin, critical evaluation of the fundamental characteristics of relaxin during pregnancy and pregnancy loss, and clarification on the strengths and limitations of relaxin as a diagnostic aid to determine pregnancy status and assess maternal-fetal health in bottlenose dolphins.

Testosterone and progesterone concentrations in blow samples are biologically relevant in belugas (Delphinapterus leucas).

Steroid hormone analysis in blow (respiratory vapor) may provide a minimally invasive way to assess the reproductive status of wild cetaceans. Biological validation of the method is needed to allow for the interpretation of hormone measurements in blow samples. Utilizing samples collected from trained belugas (Delphinapterus leucas, n=20), enzyme immunoassays for testosterone and progesterone were validated for use with beluga blow samples. Testosterone concentrations in 40 matched blood and blow samples collected from 4 male belugas demonstrated a positive correlation (R2=0.52, p<0.0001). Progesterone concentrations in 64 matching blood and blow samples from 11 females were also positively correlated (R2=0.60, p<0.0001). Testosterone concentrations (mean±SD) in blow samples collected from adult males (119.3±14.2pg/ml) were higher (p<0.01) than that of a juvenile male (<8years) (59.4±6.5pg/ml) or female belugas (54.1±25.7pg/ml). Among adult males, testosterone concentrations in blow demonstrated a seasonal pattern of secretion, with peak secretion occurring during the breeding season (February-April, 136.95±33.8pg/ml). Progesterone concentrations in blow varied by reproductive status; pregnant females (410.6±87.8pg/ml) and females in the luteal phase of the estrous cycle (339.5±51.0pg/ml) had higher (p<0.0001) blow progesterone concentrations than non-pregnant females without a corpus luteum (242.5±27.3pg/ml). Results indicate that blow sample analysis can be used to detect variation in reproductive states associated with large differences in circulating testosterone or progesterone in belugas.

Characterization and longitudinal monitoring of serum progestagens and estrogens during normal pregnancy in the killer whale (Orcinus orca).

The secretory patterns of progestagens and estrogens were characterized throughout 28 normal pregnancies until two month post-partum in eleven killer whales. HPLC analysis of serum from different reproductive stages (luteal phase, EARLY, MID, and LATE pregnancy) identified three major immunoreactive progestagen peaks; progesterone (P4), 5α-pregnane-3,20-dione (5α-DHP) and pregnanediol, with 5α-DHP approximately half of that for P4 in the luteal phase, and EARLY, but approximately 2/3 of P4 during MID and LATE pregnancy. At birth, 5α-DHP was the only significant (>10% immunoreactivity) immunoreactive progestagen detected in placental (umbilical cord) serum. Maternal recognition of pregnancy appears to occur between day 21 and 28 post-ovulation when a significant deviation in progestagen concentrations between conceptive and non-conceptive cycles was detected. Progestagen concentrations during pregnancy displayed a bimodal pattern with significant peaks (P<0.05) in EARLY (indexed month post-conception [IMPC] 2, 3, 4) and MID (IMPC 9, 10) before decreasing (P<0.05) over an 11day interval to luteal phase concentrations on the day of parturition. Among estrogens, estriol was secreted in the highest concentrations but only estrone (free and conjugated) and estradiol increased (P<0.001) during pregnancy, with peaks observed during the final month of gestation, and an influence (P<0.05) of fetal sex on estradiol production was detected. Collective findings indicate that P4 derived from the corpus luteum is the major biologically active progestagen during the luteal phase and pregnancy, and that 5α-DHP production, possibly from both luteal and placental sources, increases during the second half of pregnancy.

Characterization of estrogens, testosterone, and cortisol in normal bottlenose dolphin (Tursiops truncatus) pregnancy.

The goal of this study was to describe profiles of serum estrogens, testosterone and cortisol during normal pregnancy in bottlenose dolphins. Predominant estrogens in all categories of dolphin sera pools during estrus and pregnancy (EARLY: Days 0-120; MID: Days 121-240; LATE: Days 241 to parturition; Day 0=day of conception) were estrone/estrone conjugates (E1-C) and estriol (E3). Serum samples collected throughout 101 normal pregnancies were analyzed for E1-C, E3, testosterone (T) and cortisol (CORT). E1-C was higher (P<0.05) during LATE compared to EARLY and MID, and higher (P<0.05) in nulliparous than multiparous females. E1-C concentrations were also inversely associated with maternal age (P=0.05). E3 was higher (P<0.05) in EARLY than MID and LATE, and higher overall for nulliparous than multiparous females, but concentrations were similar among gestational stages when parity was excluded from analyses. Analysis by indexed month post-conception (IMPC) demonstrated that E1-C increased from IMPC 9 and peaked at IMPC 11. E3 was significantly elevated during IMPC 1, decreased until IMPC 6 and peaked at IMPC 11. T increased (P<0.05) at IMPC 3 and continued to increase throughout gestation (P<0.05). CORT was higher (P<0.05) during LATE compared to EARLY and MID (P<0.05), peaked during IMPC 12, and was not affected by parity. Hormone profiles were not influenced by fetal sex.

Evaluation of anti-Erysipelothrix rhusiopathiae IgG response in bottlenose dolphins Tursiops truncatus to a commercial pig vaccine.

Erysipelothrix rhusiopathiae is the causative agent of erysipeloid in humans and of erysipelas in various animals, including bottlenose dolphins Tursiops truncatus, in which an infection has the potential to cause peracute septicemia and death. The purpose of this study was to evaluate the efficacy of using an off-label porcine (ER BAC PLUS®, Zoetis) E. rhusiopathiae bactrin in a bottlenose dolphin vaccination program by determining the anti-E. rhusiopathiae antibody levels in vaccinated dolphins over a 10 yr period. Serum samples (n = 88) were analyzed using a modified fluorescent microbead immunoassay from 54 dolphins, including 3 individuals with no history of vaccination and 51 dolphins with an average of 5 vaccinations, 3 of which had previously recovered from a natural E. rhusiopathiae infection. A mean 311-fold increase in the immunoglobulin G (IgG) antibody index was measured in a subsample of 10 dolphins 14 d after the first booster vaccination. Serum IgG antibody titers were influenced by number of vaccines received (r2 = 0.47, p < 0.05) but not by age, gender, history of natural infection, adverse vaccine reaction, vaccination interval or time since last vaccination. The commercial pig bacterin was deemed effective in generating humoral immunity against E. rhusiopathiae in dolphins. However, since the probability of an adverse reaction toward the vaccine was moderately correlated (p = 0.07, r2 = 0.1) with number of vaccines administered, more research is needed to determine the optimal vaccination interval.

Chicks produced in the Magellanic penguin (Spheniscus magellanicus) after cloacal insemination of frozen-thawed semen.

The in vitro and in vivo functionality of cryopreserved spermatozoa was examined over two breeding seasons in a zoological colony of Magellanic penguins (Spheniscus magellanicus). Frozen-thawed semen was inseminated into five anesthetized females, over a total of eight egg production cycles, with a different male used for each artificial insemination (AI) within each season. Females were maintained within the colony in cordoned nest sites to prevent copulation with their paired male, and were inseminated every 3-10 days until the first oviposition. Semen frozen from seven males using a straw method retained 39.8%, 25.7%, 74.0%, and 52.1% of its initial total motility, progressive motility, average path velocity, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P < 0.05) during freeze-thawing from 76.7% immediately prior to freezing to 65.3% post-thawing. Conceptive females received 1.6 ± 0.2 inseminations before the first oviposition, with 19.2 ± 1.6 × 10(6) motile, morphologically normal spermatozoa per insemination. Overall fertility was 53.3% (8/15 eggs), hatchability was 50.0% (4/8), and genetic analyses confirmed that all embryos and hatchlings were sired by the AI male. Fertile eggs were laid at 4.0-12.1 days after AI, indicating that frozen-thawed spermatozoa resided in the female reproductive tract for up to ∼7.2 days prior to fertilization. Results demonstrate that frozen-thawed Magellanic penguin spermatozoa are fully functional in vivo and support the use of genome banking and AI as tools for managing the sustainability of zoological penguin populations. Zoo Biol. 35:326-338, 2016. © 2016 Wiley Periodicals, Inc.

MALE REPRODUCTIVE PHYSIOLOGY AND THE DEVELOPMENT OF ARTIFICIAL INSEMINATION IN THE MAGELLANIC PENGUIN (SPHENISCUS MAGELLANICUS) USING CHILLED-STORED SEMEN.

Research was performed to increase our understanding of male Magellanic penguin (Spheniscus magellanicus) reproductive biology and to develop artificial insemination (AI) technology to assist with maintaining the species' genetic diversity. Seminal traits were characterized from seven males with noncontaminated ejaculates (n = 123) displaying high in vitro motion parameters, membrane integrity, and morphology. Seven females were maintained in nest sites that permitted visual, auditory, and tactile contact with their paired male but not copulation for 18.3 ± 2.4 days before egg lay. After cloacal AI (2.6 ± 0.4 inseminations/female) with semen chilled for up to 20.5 hr at 5°C, all females produced one to two fertile eggs, with the first oviposition occurring within 7 days of plasma progesterone concentrations exceeding 0.8 ng/ml. Overall fertility was 91.7%, hatchability was 63.6%, and genetic analyses confirmed that all embryos and hatchlings were sired by AI males. The heterospermic AI design demonstrated that eggs were fertilized by spermatozoa chilled for 1.5-19.8 hr before AI and were laid 4.5-11.5 days post AI. These results contribute new data on Magellanic penguin sperm biology and demonstrate that high fertility rates after AI of chilled semen can be achieved with females remaining in proximity to their paired mate.

Semen characterization, seasonality of production, and in vitro sperm quality after chilled storage and cryopreservation in the king penguin (Aptenodytes patagonicus).

Research was conducted to examine seasonal seminal traits and to establish short-term and long-term sperm preservation methods in the king penguin (Aptenodytes patagonicus) for use in genome banking and artificial insemination (AI). Spermic ejaculates (n = 87) obtained using a cooperative method were collected across multiple (n = 6, Male 1) and a single (Male 2) breeding season(s). Non-contaminated ejaculates (n = 69) were 0.36 ± 0.32 ml at 56.3 ± 62.7 × 10(7) sperm/ml with 85.3 ± 10.6% total motility (TMot), 52.5 ± 12.9% progressive motility (PMot), 86.6 ± 24.3 µm/sec average path velocity (VAP) and 92.3 ± 3.7% plasma membrane intact. In vitro quality of chilled semen was best maintained over 48 hr at 5°C than 21°C, with decreased (P < 0.05) motility and morphology parameters observed by 24 and 6 hr, respectively. A comparison of two freezing methods (straw [STR] vs. directional [DF]) demonstrated similar effects on post-thaw quality at 0 and 3 hr, with the exception of plasma membrane integrity which was higher (P < 0.05) at 0 hr for DF (48.7 ± 6.5%) than STR (41.2 ± 7.0%). At 0 hr post-thaw, DF samples retained 46%, 69%, and 52% of their initial PMot, VAP, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P < 0.05) during freeze-thawing from 84% post-collection to 37% and 34% at 0 and 3 hr post-thaw, respectively. Results indicate that chilled and cryopreserved semen from the king penguin has potential for use in AI.