Universal DNA methylation age across mammalian tissues.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Topographic Variations in Mobilization of Blubber in Relation to Changes in Body Mass in Short-Finned Pilot Whales (Globicephala macrorhynchus).

AbstractFat-level measurements used to indicate individual body condition and fitness are useful only when taken at a region along the body where fat responds to variations in caloric intake. Investigations to identify appropriate species-specific regions are limited, especially for cetaceans that have a specialized fat (blubber) that serves as an energy reserve and provides insulation. Over 18 mo, body mass of six pilot whales varied (range: 50-172 kg), and although caloric intake increased when water temperatures were lower, generally the best-fitting state-space model for length-adjusted mass was based on a single factor, caloric intake. After correcting for body length (range: 330-447 cm), the slope for blubber thickness and "blubber ring" thickness (average blubber thickness along a girth) in relation to body mass was positive and had a P value of <0.10 at six of 16 blubber measurement sites and one of five girth measurement sites, respectively. The slope for body girth (a reflection of changes in underlying blubber thickness) in relation to body mass was positive and had a lower P value ([Formula: see text]) at three of five girth measurement sites. Results indicate that blubber from the anterior insertion of the pectoral fins to the posterior insertion of the dorsal fin is the most metabolically active region. This region includes the midflank site, a location where blubber thickness measurements have historically been taken to monitor cetacean body condition. Conversely, blubber in the peduncle region was comparatively inert. These findings must be considered when measuring blubber thickness and body width (i.e., photogrammetry) to monitor the condition of free-ranging cetaceans.

The steroid metabolome of pregnancy, insights into the maintenance of pregnancy and evolution of reproductive traits.

Modes of mammalian reproduction are diverse and not always conserved among related species. Progesterone is universally required to supports pregnancy but sites of synthesis and metabolic pathways vary widely. The steroid metabolome of mid-to late gestation was characterized, focusing on 5α-reduced pregnanes in species representing the Perissodactyla, Cetartiodactyla and Carnivora using mass spectrometry. Metabolomes and steroidogenic enzyme ortholog sequences were used in heirarchial analyses. Steroid metabolite profiles were similar within orders, whales within cetartiodactyls for instance, but with notable exceptions such as rhinoceros clustering with goats, and tapirs with pigs. Steroidogenic enzyme sequence clustering reflected expected evolutionary relationships but once again with exceptions. Human sequences (expected outgroups) clustered with perissodactyl CYP11A1, CYP17A1 and SRD5A1 gene orthologues, forming outgroups only for HSD17B1 and SRD5A2. Spotted hyena CYP19A1 clustered within the Perissodactyla, between rhinoceros and equid orthologues, whereas CYP17A1 clustered within the Carnivora. This variability highlights the random adoption of divergent physiological strategies as pregnancy evolved among genetically similar species.

Establishing models of corticosteroid patterns during the life history of killer whales (Orcinus orca) under human care.

The use of corticosteroids and their metabolites as a physiologic measure of stress in wildlife species is increasing in both in and ex situ populations. However, factors such as season, circadian rhythm, sex and age are also known to influence corticosteroid production in wildlife. Our objective was to evaluate the influence of these variables on serum cortisol, corticosterone, aldosterone and their fecal metabolites in zoo-based killer whales (Orcinus orca). For evaluation of season, sex and age, we examined 30 animals (21 females, 9 males), and for circadian rhythm, we studied 18 animals (10 females, 8 males). Season did not influence corticosteroids (P ≥ 0.19). Circadian rhythm influenced all corticosteroids (P ≤ 0.012). Serum cortisol and corticosterone were highest in the morning and lower by mid-day and evening. Serum aldosterone was lowest during mid-day and highest in the evening. Excreted corticosteroid metabolites were higher in the morning and mid-day compared to evening (P ≤ 0.002). Serum cortisol was higher in males than females (P = 0.011). The inverse was observed for fecal corticosterone metabolites (P = 0.03). Serum corticosterone increased with age in all animals (P = 0.002), but serum cortisol increased with age in males only (P < 0.001). Finally, combining previously published data and these data reported herein, we developed models of corticosteroid patterns for zoo-based killer whales during life history events, including circadian variation, pregnancy and acute stress that may serve as a guide for evaluating stress physiology and animal welfare in this species.

Anti-Müllerian hormone in managed African and Asian rhino species.

Serum collected across the lifespan of four managed rhino species: black (Diceros bicornis, n = 16), white (Ceratotherium simum simum, n = 19), greater one-horned (GOH, Rhinoceros unicornis, n = 11) and Sumatran (Dicerorhinus sumatrensis, n = 6) were validated and analyzed in an anti-Müllerian hormone (AMH) enzyme- linked immunoassay. Concentrations of AMH were examined over time, between sexes and throughout different reproductive states which included n = 3 female white rhinos immunocontracepted with porcine zona pellucida (pZP). Across species, males produced higher AMH concentrations compared to females. Among males, AMH concentrations varied by species aside from comparable values secreted between black and white rhinos. The GOH and Sumatran rhino secreted the highest and lowest male AMH concentrations, respectively. However, within each species, AMH concentrations were similar across male age categories. Preliminary insight into male AMH changes from birth to sexual maturity suggest its potential as a marker for onset of testicular maturation. Female black, GOH and Sumatran rhinos secreted comparable AMH concentrations which were higher than those in white rhino. Within each species, inter-individual variation in AMH secretion occurred among females of similar age. While AMH secretion did not differ across the ages sampled for female white (4->26 yr) and GOH (4-26 yr) rhinos, black and Sumatran rhinos >26 and <4 yr, respectively secreted lower AMH compared to conspecific females 7-26 yr of age. Two idiopathic infertility cases corresponded to low (outside species range) AMH values. The establishment of normative AMH concentrations in managed African and Asian rhinos provides an additional metric beyond traditional sex steroids to assess gonadal function. Further work is needed to determine if AMH can predict fertility potential in rhinos.

Thyroid hormone concentrations associated with age, sex, reproductive status and apparent reproductive failure in the Amazon river dolphin (Inia geoffrensis).

This study was conducted to characterize immunoreactive thyroid hormone concentrations in wild Amazon river dolphins, also called boto (Inia geoffrensis) by age group, sex, pregnancy and lactation status, and to determine if thyroid hormone concentration differences could be detected between pregnant females with and without successful parturition outcomes. Radioimmunoassays were used to analyse total T 3 and total T 4 in 182 serum samples collected from 172 botos living in the Mamirauá Sustainable Development Reserve, in the Brazilian Amazon from 2003 through 2015. Age significantly affected tT 3 and tT 4 concentrations in males, with values in immature males and females being significantly lower than those in adult males, whereas no age effects were noted between immature females and adult non-pregnant, non-lactating females. Significant sex differences were noted in tT 3 concentrations between immature males and females and in tT 4 concentrations between adult males and females. These resulted in significant differences in the tT 3:tT 4 ratio between males and females within the immature and adult groups. Lactating and non-pregnant adult females had significantly higher tT 3 concentrations than pregnant females, and this difference was primarily driven by a 12% drop in tT 3 concentrations during the last two-thirds of pregnancy. No differences in thyroid hormone concentrations were detected between females diagnosed as pregnant and later found to have or not have a live calf. These results are the first to define thyroid hormone reference intervals and normal physiological variations in a wild population of river dolphins.

Androgen and glucocorticoid production in the male killer whale (Orcinus orca): influence of age, maturity, and environmental factors.

Circulating concentrations of testosterone and its precursor androstenedione, as well as dehydroepiandrosterone (DHEA) and the adrenal hormones cortisol and corticosterone were measured at monthly intervals in 14 male killer whales (Orcinus orca) aged 0.8-38 years. Analyses were performed for examination of the relationships of age, sexual maturation status (STATUS), season, and environmental temperature (monthly air ambient temperature, A-TEMP) with hormone production using a mixed effects linear regression model with animal ID as the random variable. Hormone profiles, derived from enzyme immunoassay procedures validated herein, established that simultaneous up-regulation of androstenedione and testosterone production occurs at puberty, when males are aged 8-12 years. Androgen (testosterone and androstenedione) production in pubertal and adult males was influenced by season, with highest (p < 0.01) concentrations observed in spring and summer months. A significant effect of STATUS and season on DHEA production was also documented, with higher (p < 0.05) concentrations in pubertal and adult males compared to juvenile males, and higher (p < 0.05) concentrations in the months of summer than the fall. Among adult males (≥13 years), those classified as aged (≥31 years) had concentrations of testosterone and both glucocorticoids that were lower (p < 0.05), and those of androstenedione that were higher (p < 0.05) than their younger counterparts. The cortisol:corticosterone ratio for adult males was 7 : 1, and both glucocorticoids were affected by STATUS (p < 0.05), but not season or A-TEMP. Results of this research enhance our understanding of reproductive and adrenocortical function in healthy male killer whales and provide baseline profiles of hormone production for use in the species' health assessment and conservation.

Factors impacting the success of post-mortem sperm rescue in the rhinoceros.

The goal of this study was to identify factors that influenced the ability to successfully rescue sperm post-mortem from rhinoceroses maintained in North American zoos. Factors considered included procedural technicalities, individual rhinoceros characteristics and timing. Gross testicular pathology was noted in 17.4% of males (4/23) but did not impact sperm recovery except in one case of azoospermia (4.3%). Of the males in which sperm recovery was attempted (n=21), 62% yielded quality samples considered adequate for cryopreservation (≥ 30% motility with ≥ 2.0 forward progressive status). A high percentage of males (70.6%; 12/17) from which reproductive tissue was removed an d cooled ≤ 4 h after death yielded quality sperm samples, whereas only 25% (1/4) of males from which tissue was removed>4h after death yielded quality samples. Quality samples were recovered 1-51 h post-mortem from rhinoceroses 8 to 36 years old. Neither type of illness (prolonged or acute), or method of death (euthanasia or natural) affected the ability to harvest quality samples (P > 0.05). The Indian rhinoceros yielded significantly more sperm on average (40 × 10(9)) than the African black rhinoceros (3.6 × 10(9); P < 0.01) and the African white rhinoceros (3.2 × 10(9); P < 0.05). Across all species and samples assessed (n = 11), mean post-thaw sperm motility (41%), was only 15% less than pre-freeze motility (56%) and only decreased to 22% during the 6h post-thaw assessment period. Rhinoceros sperm rescue post-mortem is relatively successful across a wide range of variables, especially when tissues are removed and cooled promptly after death, and should be considered standard practice among zoos.

Reproductive physiology of the female Magellanic penguin (Spheniscus magellanicus): Insights from the study of a zoological colony.

Eight captive female Magellanic penguins (Spheniscus magellanicus) were monitored over a 10week period, commencing at 5weeks prior to egg lay (EL), to increase our understanding of the species' reproductive biology. Females in cordoned nest sites underwent cloacal artificial insemination (AI) every 4-7days with different semen donors for each insemination. The EL interval was 97.9±3.6h (range: 84-108h) and paternity analyses revealed that conceptive inseminations occurred from 11.5 to 4.5days before oviposition. A biphasic pattern of estradiol, testosterone, progesterone and the biochemical analytes triglyceride, iron, calcium and phosphorus occurred in relation to EL, with values increasing (P<0.05) to maximal concentrations during the three weeks preceding oviposition, then decreasing (P<0.05) rapidly after oviposition completion. In comparison with post-lay (baseline) values, concentrations of estradiol and testosterone relative to the first oviposition were elevated at Week-5, and those of triglyceride, a yolk formation index, as well as iron, calcium and phosphorus, became elevated at Week-4 (P<0.05). Collective data indicate an estimated total egg formation interval of 29days, with oviducal transit of the ovulated ovum occurring over the majority of the ∼4day EL interval. These findings indicate that egg formation is prolonged with folliculogenesis initiated at 5weeks or more prior to oviposition. Consequently, the period of folliculogenesis and egg formation is estimated to overlap with the final ∼3weeks that wild females spend at sea prior to returning to land for breeding.

Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum).

White rhinoceros ejaculates (n=9) collected by electroejaculation from four males were shipped (10°C, 12h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0µg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5°C up to 54h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54h post-collection, some motility parameters were higher (P<0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P<0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89±6%) or Y-bearing (86±3%) spermatozoa was achieved using moderate sorting rates (2540±498X-spermatozoa/s; 1800±557Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI.

Sperm DNA fragmentation and morphological degeneration in chilled elephant (Elephas maximus and Loxodonta Africana) semen collected by transrectal massage.

Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0 h; subjective motility parameters only) and after 24 h of chilled storage at 10 °C (24 h; all ejaculate and sperm characteristics). Ejaculates with ≤50% total motility (TM) at 24 h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.2 ± 19.2% fragmented DNA) and an elevated incidence of detached heads (43.3 ± 22.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24 h displayed higher (p < 0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48 h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24 h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant.

Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing.

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6±6.0% progressive motility, 3.9±1.5 µg creatinine mL(-1) raw semen, 2.7±0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P>0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P<0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P<0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P>0.05) across all groups and processing stages (1.6±0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

The relationship of maternal characteristics and circulating progesterone concentrations with reproductive outcome in the bottlenose dolphin (Tursiops truncatus) after artificial insemination, with and without ovulation induction, and natural breeding.

Bottlenose dolphins (Tursiops truncatus) undergoing natural breeding and artificial insemination (AI) were examined to characterize serum progesterone concentrations and determine relationships among age, parity, and reproductive outcome. Progesterone profiles of five cycle types (n = 119 total cycles from 54 animals) were characterized as follows: (i) conception and production of a live term calf (conceptive-term, n = 73); (ii) conception and abortion after Day 60 (conceptive-abortion, n = 12); (iii) unknown conception status with prolonged, elevated progesterone and absence of a fetus (conceptive-unknown, n = 14); (iv) conception failure with normal luteal phase progesterone concentrations (non-conceptive, n = 14, AI cycles only); and (v) conception failure with progesterone insufficiency occuring after spontaneous ovulation or owing to premature ovulation induction using GnRH (non-conceptive-PI, n = 6, AI cycles only). By Day 21 post-insemination (PI), progesterone concentrations were similar (P > 0.05) among conceptive-term, conceptive-abortion and conceptive-unknown, and higher (P < 0.05) for conceptive-term than non-conceptive and non-conceptive-PI cycles. Progesterone concentrations of known conceptive cycles peaked by Week 7 PI (P < 0.05) and remained elevated for the remainder of pregnancy (Weeks 8 up to 54, ≥ 5 days pre-partum). During midpregnancy (Days 121-240), conceptive-term cycles had higher (P > 0.05) progesterone concentrations than conceptive-abortion and unknown conception status cycles. Parity was not associated with reproductive outcome based on cycle type (P > 0.05). Age of females in conceptive-unknown (26.5 ± 10.1 yrs) and conceptive-abortion (22.1 ± 9.4 yrs) groups was higher (P < 0.05) than in conceptive-term (15.7 ± 7.2 yrs). The conceptive-unknown cycle type possibly represents undetected early embryonic loss occurring before Day 60 PI. Length of gestation using known conception dates was 376.1 ± 11.0 days and the range of this parameter (355-395 days) has implications for peri-parturient management procedures for the species.

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Characterization of the estrous cycle in female beluga (Delphinapterus leucas) using urinary endocrine monitoring and transabdominal ultrasound: Evidence of facultative induced ovulation.

Recent, successful application of assisted reproductive technologies in captive beluga has resulted from the extensive study of male beluga reproductive biology. Optimization of assisted reproduction requires additional detailed knowledge of the female estrous cycle. Our specific objectives were to: (1) validate urinary immunoassays for use in this species; (2) elucidate annual ovarian cycle dynamics through the combined use of hormone excretion patterns and transabdominal ultrasound; and (3) establish whether ovulation in this species is spontaneous or induced by male factors. Ovulation was observed in four of 15 estrous cycles monitored in four adult female beluga maintained in a single-sex group. After introduction of a breeding male, ovulation was observed in six of seven estrous cycles. All estrous cycles occurred from March through June. For spontaneous ovulations (n=4), the inter-estrous interval was 34d (range 33-35d), with a follicular phase length (FPL) of 25±8d (mean±SD). For all ovulatory estrous cycles (with and without a breeding male), urinary estrogen conjugates (EC, 15.3±7.9ng/mg Cr) and ovulatory luteinizing hormone (ovLH, 17.1±6.6ng/mg Cr) concentrations both peaked on Day 0, and EC concentrations returned to baseline 8±7d later. For non-conceptive cycles, urinary progestagen (Pg) concentrations increased on Day 0 (3.5±1.7ng/mg Cr), peaked on Day+19 (19.7±17.1ng/mg Cr), and were elevated above baseline for 27±4d. Preovulatory follicular diameter and circumference on Day -2±2 (range: Day -4 to -1) from peak EC were 2.5±0.7 and 7.8±1.3cm, respectively. The FPL in non-ovulatory estrous cycles (n=11) lasted 24±10d and EC concentrations gradually declined to baseline over a 21±10d interval following the EC peak (27.8±28.8ng/mg Cr). Non-ovulatory estrous cycles were characterized by the absence of an ovLH surge and no concomitant increase in Pg concentrations above baseline excretion; the mean follicular diameter at or near peak EC was 3.1±0.8cm on Day 2 ±3d from peak EC (range: -1 to +5days from peak EC). Overall, these data confirm that captive beluga exhibit reproductive seasonality and demonstrate that the species is a facultative-induced ovulator.

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca).

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10(6) sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.

Alkaline phosphatase as an indicator of true ejaculation in the rhinoceros.

The objective was to determine if seminal alkaline phosphatase (ALP) can serve as an indicator of true ejaculation in the rhinoceros. Concentrations of ALP activity were determined in seminal fractions collected from African black rhinos (Diceros bicornis), an African white rhino (Ceratotherium simum), and an Indian rhino (Rhinoceros unicornis) during electroejaculation. In addition, seminal fractions collected during penile massage of a Sumatran rhino (Dicerorhinus sumatrensis) were assessed. Correlations between ALP activity and sperm concentration, fraction pH, and fraction osmolality were evaluated in the Indian rhino and black rhino. Concentrations of ALP activity in rhino ejaculate fractions ranged from < 5 to 11,780 U/L and were positively correlated (P < 0.05) with sperm concentration for both Indian rhino (r = 0.995) and black rhino (r = 0.697), but did not exhibit a strong correlation with either pH or osmolality (P > 0.05). Data were insufficient for establishing meaningful correlation coefficients in the Sumatran rhino and white rhino, but preliminary results were in accordance with findings in the Indian rhino and black rhino. We concluded that ALP was present in rhinoceros semen, likely originated from the epididymides and/or testes, and could serve as a useful tool for assessing the production of ejaculatory versus pre-ejaculatory fluid in the rhinoceros.

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.

Preservation of beluga (Delphinapterus leucas) spermatozoa using a trehalose-based cryodiluent and directional freezing technology.

A beluga (Delphinapterus leucas) sperm preservation method was developed for use in genome banking and AI. In Study 1, glycerol-based cryodiluents (modified BF5F and modified Platz Diluent Variant (PDV)) were unable to maintain adequate progressive motility using straws (fast and slow freezing rate (FR)) or pellets (slow FR). Neither freezing method nor FR affected in vitro sperm characteristics (P > 0.05), but retention of prefreeze progressive motility following thawing was greater (P < 0.05) for BF5F (21%) than PDV (15%). In Study 2, examining the effects of straw freeze-thawing using BF5F with glycerol (1 and 3%, v/v) or trehalose (46 and 91 mM) on sperm characteristics, samples cryopreserved in trehalose exhibited superior (P < 0.05) in vitro parameters compared with their glycerol-treated counterparts. In Study 3, compared with a straw method, directional freezing using 91 mM trehalose enhanced (P < 0.05) sperm characteristics, with samples retaining 38%, 75% and 61% of their prefreeze progressive motility, curvilinear velocity and viability, respectively. A higher (P < 0.05) proportion of motile spermatozoa displayed rapid velocity after directional (21 +/- 1%) compared with straw (12 +/- 3%) freezing. Systematic development of a cryodiluent and the use of directional freezing resulted in beluga spermatozoa exhibiting adequate post-thaw quality for genome banking and use in AI.