Secretory Pattern of Inhibin During Estrous Cycle and Pregnancy in African (Loxodonta africana) and Asian (Elephas maximus) Elephants

The ovary of female elephants has multiple corpora lutea (CL) during the estrous cycle and gestation. The previous reports clearly demonstrated that inhibin was secreted from lutein cells as well as granulosa cells of antral follicles in cyclic Asian elephants. The aim of this study is to investigate the inhibin secretion during the pregnancy in African and Asian elephants. Two African elephants and two Asian elephants were subjected to this study. Circulating levels of immunoreactive (ir‐) inhibin and progesterone were measured by radioimmunoassay. Four pregnant periods of an African elephant and three pregnant periods of an Asian elephant were analyzed in this study. Circulating levels of ir‐inhibin started to increase at 1 or 2 week before the ovulation and reached the peak level 3 or 4 weeks earlier than progesterone during the estrous cycle in both African and Asian elephants. After last luteal phase, the serum levels of ir‐inhibin remained low throughout pregnancy in both an African and an Asian elephant. The mean levels of ir‐inhibin during the pregnancy were lower than the luteal phase in the estrous cycle despite high progesterone levels were maintained throughout the pregnancy. These results strongly suggest that CL secrete a large amount of progesterone but not inhibin during the pregnancy in elephants. Zoo Biol 31:511‐522, 2012. © 2011 Wiley Periodicals, Inc.     

Survey of the reproductive cyclicity status of Asian and African elephants in North America

The Asian and African elephant populations in North America are not self‐sustaining, and reproductive rates remain low. One problem identified from routine progestagen analyses is that some elephant females do not exhibit normal ovarian cycles. To better understand the extent of this problem, the Elephant TAG/SSP conducted a survey to determine the reproductive status of the captive population based on hormone and ultrasound evaluations. The survey response rates for facilities with Asian and African elephants were 81% and 71%, respectively, for the studbook populations, and nearly 100% for the SSP facilities. Of the elephants surveyed, 49% of Asian and 62% of African elephant females were being monitored for ovarian cyclicity via serum or urinary progestagen analyses on a weekly basis. Of these, 14% of Asian and 29% of African elephants either were not cycling at all or exhibited irregular cycles. For both species, ovarian inactivity was more prevalent in the older age categories (>30 years); however, acyclicity was found in all age groups of African elephants. Fewer elephant females (～30%) had been examined by transrectal ultrasound to assess reproductive‐tract integrity, and corresponding hormonal data were available for about three‐quarters of these females. Within this subset, most (～75%) cycling females had normal reproductive‐tract morphologies, whereas at least 70% of noncycling females exhibited some type of ovarian or uterine pathology. In summary, the survey results suggest that ovarian inactivity is a significant reproductive problem for elephants held in zoos, especially African elephants. To increase the fecundity of captive elephants, females should be bred at a young age, before reproductive pathologies occur. However, a significant number of older Asian elephants are still not being reproductively monitored. More significantly, many prime reproductive‐age (10–30 years) African females are not being monitored. This lack of information makes it difficult to determine what factors affect the reproductive health of elephants, and to develop mitigating treatments to reinitiate reproductive cyclicity. Zoo Biol 23:309–321, 2004. © 2004 Wiley‐Liss, Inc.     

Secretory patterns of serum prolactin in Asian (Elephas maximus) and African (Loxodonta africana) elephants during different reproductive states: Comparison with concentrations in a noncycling African elephant

Serum prolactin was quantified in adult female Asian (Elephas maximus) and African (Loxodonta africana) elephants during various reproductive states and the profiles compared to that in a noncycling African elephant. In reproductively normal elephants, there was no effect of season, estrous cycle stage, or lactational status on quantitative or qualitative prolactin secretion (P > 0.05), nor where there any differences (P > 0.05) in overall prolactin concentrations between species. In pregnant elephants, prolactin concentrations remained at baseline for the first 4–6 months of gestation. Thereafter, concentrations during early pregnancy averaged ∼four-fold higher than those during the estrous cycle, increasing to ∼100-fold over baseline during mid- to late gestation in both species. In contrast to cycling elephants, prolactin concentrations in an African elephant exhibiting chronic anovulation (on the basis of an acyclic serum progesterone profile) and mild galactorrhea were consistently about five-fold higher (P < 0.05), suggesting she is hyperprolactinemic. Other endocrinological assesments confirmed the hypogonadal state of this female. Serum estradiol concentrations were consistently at or below detectable levels. Additionally, no preovulatory luteinizing hormone (LH) surges occurred in daily serum samples analyzed over a 12-month period. The pituitary was not totally refractory, however, and responded with a several-fold increase in serum LH concentration (peak, 3.07 ng/ml) over baseline (0.75 ng/ml) after i.v. injection of gonadotropin-releasing hormone. This study describes normal baseline serum prolactin values for Asian and African elephants and is the first to identify hyperprolactinemia as a possible cause of reproductive acyclicity and galactorrhea in an African elephant. Zoo Biol 16:149–159, 1997. © 1997 Wiley-Liss, Inc.     

Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses

In the present work we have studied in Neospra caninum aborted bovine foetuses the influence of foetal age (first, second and third gestational periods) on parasite distribution by nested PCR, parasite loads by real-time PCR and N. caninum associated lesions. For this purpose, a total of 220 aborted foetuses were analysed and detection of N. caninum infection was accomplished by nested-PCR in brain, heart and liver, detecting the presence of the parasite in 72 (32.7%) bovine foetuses. When the different age classes were compared, parasite DNA-detectability in heart and liver was reduced over time of gestation (P < 0.05, Fisher F-test). N. caninum distribution, parasite loads and lesions were studied on 34 out of 72 N. caninum-infected foetuses selected according to the stage of pregnancy and organs recovered. A higher number of positive-PCR tissue samples were observed in the foetuses corresponding to the first and second pregnancy periods. In the last trimester, the parasite could only be detected in the brain and, sporadically, in the diaphragm, heart and lymph nodes. The parasite loads decreased during pregnancy and the foetuses from the first period had higher parasite burdens in brain, heart, kidney and lung (P < 0.05, Kruskal-Wallis H-test) than in those corresponding to the other two trimesters of pregnancy. In addition, the observed lesions were more severe in foetuses from the first and second pregnancy periods than those from the third period (P > 0.05, Kruskal-Wallis H-test). Our results confirm the influence of N. caninum foetal age on pathogenesis in natural N. caninum infections.     

Elephant Genomes Reveal Accelerated Evolution in Mechanisms Underlying Disease Defenses

Disease susceptibility and resistance are important factors for the conservation of endangered species, including elephants. We analyzed pathology data from 26 zoos and report that Asian elephants have increased neoplasia and malignancy prevalence compared with African bush elephants. This is consistent with observed higher susceptibility to tuberculosis and elephant endotheliotropic herpesvirus (EEHV) in Asian elephants. To investigate genetic mechanisms underlying disease resistance, including differential responses between species, among other elephant traits, we sequenced multiple elephant genomes. We report a draft assembly for an Asian elephant, and defined 862 and 1,017 conserved potential regulatory elements in Asian and African bush elephants, respectively. In the genomes of both elephant species, conserved elements were significantly enriched with genes differentially expressed between the species. In Asian elephants, these putative regulatory regions were involved in immunity pathways including tumor-necrosis factor, which plays an important role in EEHV response. Genomic sequences of African bush, forest, and Asian elephant genomes revealed extensive sequence conservation at TP53 retrogene loci across three species, which may be related to TP53 functionality in elephant cancer resistance. Positive selection scans revealed outlier genes related to additional elephant traits. Our study suggests that gene regulation plays an important role in the differential inflammatory response of Asian and African elephants, leading to increased infectious disease and cancer susceptibility in Asian elephants. These genomic discoveries can inform future functional and translational studies aimed at identifying effective treatment approaches for ill elephants, which may improve conservation.     

Epigenetic clock and methylation studies in elephants

Age‐associated DNA‐methylation profiles have been used successfully to develop highly accurate biomarkers of age ("epigenetic clocks") in humans, mice, dogs, and other species. Here we present epigenetic clocks for African and Asian elephants. These clocks were developed using novel DNA methylation profiles of 140 elephant blood samples of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We present epigenetic clocks for Asian elephants (Elephas maximus), African elephants (Loxodonta africana), and both elephant species combined. Two additional human‐elephant clocks were constructed by combining human and elephant samples. Epigenome‐wide association studies identified elephant age‐related CpGs and their proximal genes. The products of these genes play important roles in cellular differentiation, organismal development, metabolism, and circadian rhythms. Intracellular events observed to change with age included the methylation of bivalent chromatin domains, and targets of polycomb repressive complexes. These readily available epigenetic clocks can be used for elephant conservation efforts where accurate estimates of age are needed to predict demographic trends.     

Seasonality of reproduction in Asian elephants Elephas maximus and African elephants Loxodonta africana: underlying photoperiodic cueing?

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Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants

To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.     

Observations on the length of the intestinal tract of African <Emphasis Type="Italic">Loxodonta africana</Emphasis> (Blumenbach 1797) and Asian elephants <Emphasis Type="Italic">Elephas maximus</Emphasis> (Linné 1735)

The digestive tract of elephants is surprisingly short compared to other herbivorous mammals. However, measurements relating the length of the intestine to the body mass of the respective individual are rare. In this study, we report such data for an African elephant and an Asian elephant. Our data support the hypothesis that Asian elephants have a longer intestinal tract than their African counterparts. These findings are in accord with the observation of longer retention times and higher digestion coefficients in Asian as compared to African elephants. This difference between the species could be the reflection of slightly different ecological niches, with Asian elephants adapted to a natural diet with a higher proportion of grass.     

Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild

The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10⁶ cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for genetic diversity management and phenotype selection in these endangered mammals.     

Ovarian function in the elephant: luteinizing hormone and progesterone cycles in African and Asian elephants

Serum samples were collected weekly for 3 yr from two female African elephants, for 18 mo from two other female African elephants, and for 2 yr from two female Asian elephants. Animals were not sedated at the time of blood collection. Ovarian cycles, characterized by changes in progesterone and immunoreactive luteinizing hormone (ILH) concentrations, averaged 15.9 +/- 0.6 wk (N = 25) for African females and 14.7 +/- 0.5 wk for Asian females (N = 10). The length of the active luteal phase averaged 10.0 +/- 0.3 wk for African elephants (range 8-14 wk) and 10.6 +/- 0.6 wk for Asian females (range 9-13 wk). Interluteal phases were 5.9 +/- 0.6 wk for African females and 4.2 +/- 0.5 wk for Asian females. One African female (Maliaca) had two extended interluteal phases, both occurring between the months of February and May. Excluding these two periods, there were no differences in the length of the ovarian cycle or the length of the luteal phase between species of elephant. Serum progesterone in both species ranged from less than 50 pg/ml to 933 pg/ml. Average progesterone concentrations during the luteal phase were significantly lower in African elephants compared with Asian elephants (328 +/- 13, N = 30 cycles vs. 456 +/- 23, N = 14 cycles; p less than 0.001). ILH ranged from nondetectable to 11.6 ng/ml. These data suggest that the length of the ovarian cycle in the African elephant is about 16 wk and confirm that the length of the ovarian cycle in the Asian elephant is about 15 wk.     

Molecular and morphological evidence on the phylogeny of the Elephantidae

The African and Asian elephants and the mammoth diverged ca. 4-6 million years ago and their phylogenetic relationship has been controversial. Morphological studies have suggested a mammoth Asian elephant relationship, while molecular studies have produced conflicting results. We obtained cytochrome b sequences of up to 545 base pairs from five mammoths, 14 Asian and eight African elephants. A high degree of polymorphism is detected within species. With a dugong sequence used as the outgroup, parsimony and maximum-likelihood analyses support a mammoth African elephant clade. As the dugong is a very distant outgroup, we employ likelihood analysis to root the tree with a molecular clock, and use bootstrap and Bayesian analyses to quantify the relative support for different topologies. The analyses support the mammoth African elephant relationship, although other trees cannot be rejected. Ancestral polymorphisms may have resulted in gene trees differing from the species phylogeny Examination of morphological data, especially from primitive fossil members, indicates that some supposed synapomorphies between the mammoth and Asian elephant are variable, others convergent or autapomorphous. A mammoth African elephant relationship is not excluded. Our results highlight the need, in both morphological and molecular phylogenetics, for multiple markers and close attention to within-taxon variation and outgroup selection.     

The historical development of zoo elephant survivorship

In the discussion about zoo elephant husbandry, the report of Clubb et al. (2008, Science 322: 1649) that zoo elephants had a “compromised survivorship” compared to certain non-zoo populations is a grave argument, and was possibly one of the triggers of a large variety of investigations into zoo elephant welfare, and changes in zoo elephant management. A side observation of that report was that whereas survivorship in African elephants (Loxodonta africana) improved since 1960, this was not the case in Asian elephants (Elephas maximus). We used historical data (based on the Species360 database) to revisit this aspect, including recent developments since 2008. Assessing the North American and European populations from 1910 until today, there were significant improvements of adult (≥10 years) survivorship in both species. For the period from 1960 until today, survivorship improvement was significant for African elephants and close to a significant improvement in Asian elephants; Asian elephants generally had a higher survivorship than Africans. Juvenile (<10 years) survivorship did not change significantly since 1960 and was higher in African elephants, most likely due to the effect of elephant herpes virus on Asian elephants. Current zoo elephant survivorship is higher than some, and lower than some other non-zoo populations. We discuss that in our view, the shape of the survivorship curve, and its change over time, are more relevant than comparisons with specific populations. Zoo elephant survivorship should be monitored continuously, and the expectation of a continuous trend towards improvement should be met.     

Milk intake and development in a newborn captive African elephant (Loxodonta africana)

In August 2003 the San Diego Zoo's Wild Animal Park (WAP) and the Lowry Park Zoo, under the auspices of the AZA's Elephant SSP and a USFWS permit, imported 3.8 African elephants (Loxodonta africana) from the Kingdom of Swaziland. When they were captured, transrectal ultrasound examinations revealed that one nulliparous cow was approximately 10 months pregnant. At the time of their arrival (August 2003), all of the animals were estimated to be approximately 13 years old and were thought to be nulliparous. Based on the ultrasound examination results and the average African elephant gestation period, parturition was predicted to be 20 February 2004. In this report, we provide the first detailed data about nursing activity around the clock and newborn calf development, describe maternal and neonatal nighttime activity budgets, and explore maternal weight changes during suckling and lactation. The newborn calf suckled significantly more at night than during the day, but suckled for only about 2 hr per 24‐hr period. Regression analysis revealed that through the first 3 months of life the calf gained 0.385 kg/day while it suckled on a regular basis. We compare our findings with published information on wild elephants, and conclude that although the growth rate is reduced compared to hand‐reared elephant calves, the suckling patterns are almost identical to those reported for wild calves. Zoo Biol 0:1–7, 2005. © 2005 Wiley‐Liss, Inc.     

象科动物对当地森林生态系统的影响

现存的象科动物(Elephantidae)分为非洲草原象(Loxodonta africana)、非洲森林象(Loxodonta cyclotis)和亚洲象(Elephas maximus)3 种,作为森林生态系统的关键物种,它们对当地森林生态系统的影响非常复杂,在一定环境条件下,既可能是积极的作用,也可能是消极的作用。积极的作用包括:帮助植物传播种子;促进种子萌发;创造断层,维持群落多样性;为其他动物增加食物资源;为其他动物创造栖息地。消极的作用包括:使一些物种的种群数量减小;使森林变成灌木丛和草原等。而由于活动受限导致的种群密度过高是象科动物对森林生态系统产生消极作用的主要原因。当前象科动物的3 个物种均面临种群数量锐减和生存空间不断缩小的危机,为此迫切需要针对其对生物多样性和生态平衡的影响开展深入和全面的研究,并且应根据实际情况因地制宜地制定管理措施。     

Pathogenic copy number variations are associated with foetal short femur length in a tertiary referral centre study

Shortened foetal femur length (FL) is a common abnormal phenotype that often causes anxiety in pregnant women, and standard clinical treatments remain unavailable. We investigated the clinical characteristics, genetic aetiology and obstetric pregnancy outcomes of foetuses with short FL and provided a reference for perinatal management of such cases. Chromosomal microarray analysis was used to analyse the copy number variations (CNV) in short FL foetuses. Of the 218 foetuses with short FL, 33 foetuses exhibited abnormal CNVs, including 19 with pathogenic CNVs and 14 with variations of uncertain clinical significance. Of the 19 foetuses with pathogenic CNVs, four had aneuploidy, 14 had deletions/duplications, and one had pathogenic uniparental diploidy. The 7q11.23 microdeletion was detected in three foetuses. The severity of short FL was not associated with the rate of pathogenic CNVs. The duration of short FL for the intrauterine ultrasound phenotype in foetuses carrying a pathogenic CNV was independent of the gestational age. Further, maternal age was not associated with the incidence of foetal pathogenic CNVs. Adverse pregnancy outcomes occurred in 77 cases, including termination of pregnancy in 63 cases, postnatal dwarfed foetuses with intellectual disability in 11 cases, and three deaths within 3 months of birth. Pathogenic CNVs closely related to foetal short FL were identified, among which the 7q11.23 microdeletion was highly associated with short FL development. This study provides a reference for the perinatal management of foetuses with short FL.     

Genomic inferences from Afrotheria and the evolution of elephants

Recent genetic studies have established that African forest and savanna elephants are distinct species with dissociated cytonuclear genomic patterns, and have identified Asian elephants from Borneo and Sumatra as conservation priorities. Representative of Afrotheria, a superordinal clade encompassing six eutherian orders, the African savanna elephant was among the first mammals chosen for whole-genome sequencing to provide a comparative understanding of the human genome. Elephants have large and complex brains and display advanced levels of social structure, communication, learning and intelligence. The elephant genome sequence might prove useful for comparative genomic studies of these advanced traits, which have appeared independently in only three mammalian orders: primates, cetaceans and proboscideans.     

PHYLOGEOGRAPHY OF THE ASIAN ELEPHANT (ELEPHAS MAXIMUS) BASED ON MITOCHONDRIAL DNA

Abstract Populations of the Asian elephant (Elephas maximus) have been reduced in size and become highly fragmented during the past 3000 to 4000 years. Historical records reveal elephant dispersal by humans via trade and war. How have these anthropogenic impacts affected genetic variation and structure of Asian elephant populations? We sequenced mitochondrial DNA (mtDNA) to assay genetic variation and phylogeography across much of the Asian elephant's range. Initially we compare cytochrome b sequences (cyt b) between nine Asian and five African elephants and use the fossil‐based age of their separation (～5 million years ago) to obtain a rate of about 0.013 (95% CI = 0.011–0.018) corrected sequence divergence per million years. We also assess variation in part of the mtDNA control region (CR) and adjacent tRNA genes in 57 Asian elephants from seven countries (Sri Lanka, India, Nepal, Myanmar, Thailand, Malaysia, and Indonesia). Asian elephants have typical levels of mtDNA variation, and coalescence analyses suggest their populations were growing in the late Pleistocene. Reconstructed phylogenies reveal two major clades (A and B) differing on average by HKY85/Γ‐corrected distances of 0.020 for cyt b and 0.050 for the CR segment (corresponding to a coalescence time based on our cyt b rate of ～1.2 million years). Individuals of both major clades exist in all locations but Indonesia and Malaysia. Most elephants from Malaysia and all from Indonesia are in well‐supported, basal clades within clade A, thus supporting their status as evolutionarily significant units (ESUs). The proportion of clade A individuals decreases to the north, which could result from retention and subsequent loss of ancient lineages in long‐term stable populations or, perhaps more likely, via recent mixing of two expanding populations that were isolated in the mid‐Pleistocene. The distribution of clade A individuals appears to have been impacted by human trade in elephants among Myanmar, Sri Lanka, and India, and the subspecies and ESU statuses of Sri Lankan elephants are not supported by molecular data.     

Successful artificial insemination of an Asian elephant at the National Zoological Park

For decades, attempts to breed elephants using artificial insemination (AI) have failed despite considerable efforts and the use of various approaches. However, recent advances in equipment technology and endocrine‐monitoring techniques have resulted in 12 elephants conceiving by AI within a 4‐year period (1998–2002). The successful AI technique employs a unique endoscope‐guided catheter and transrectal ultrasound to deliver semen into the anterior vagina or cervix, and uses the “double LH surge” (i.e., identifying the anovulatory LH (anLH) surge that predictably occurs 3 weeks before the ovulatory LH (ovLH) surge to time insemination. This study describes the 6‐year collaboration between the National Zoological Park (NZP) and the Institute for Zoo Biology and Wildlife Research (IZW), Berlin, Germany, that led to the refinement of this AI technique and subsequent production of an Asian elephant calf. The NZP female was the first elephant to be inseminated using the new AI approach, and was the fifth to conceive. A total of six AI trials were conducted beginning in 1995, and conception occurred in 2000. Semen was collected by manual rectal stimulation from several bulls in North America. Sperm quality among the bulls was variable and was thus a limiting factor for AI. For the successful AI, semen quality was good to excellent (75–90% motile sperm), and sperm was deposited into the anterior vagina on the day before and the day of the ovLH surge. Based on transrectal ultrasound, ovulation occurred the day after the ovLH surge. Pregnancy was monitored by serum and urinary progestagen, and serum prolactin analyses in samples collected weekly. Fetal development was assessed at 12, 20, and 28 weeks of gestation using transrectal ultrasound. Elevated testosterone measured in the maternal circulation after 36 weeks of gestation reliably predicted the calf was a male. Parturition was induced by administration of 40 IU oxytocin 3 days after serum progestagens dropped to undetectable baseline levels. We conclude that AI has potential as a supplement to natural breeding, and will be invaluable for improving the genetic management of elephants, provided that problems associated with inadequate numbers of trained personnel and semen donors are resolved. Zoo Biol 23:45–63, 2004. © 2004 Wiley‐Liss, Inc.