Skeletal development in the African elephant and ossification timing in placental mammals.

We provide here unique data on elephant skeletal ontogeny. We focus on the sequence of cranial and post-cranial ossification events during growth in the African elephant (Loxodonta africana). Previous analyses on ossification sequences in mammals have focused on monotremes, marsupials, boreoeutherian and xenarthran placentals. Here, we add data on ossification sequences in an afrotherian. We use two different methods to quantify sequence heterochrony: the sequence method and event-paring/Parsimov. Compared with other placentals, elephants show late ossifications of the basicranium, manual and pedal phalanges, and early ossifications of the ischium and metacarpals. Moreover, ossification in elephants starts very early and progresses rapidly. Specifically, the elephant exhibits the same percentage of bones showing an ossification centre at the end of the first third of its gestation period as the mouse and hamster have close to birth. Elephants show a number of features of their ossification patterns that differ from those of other placental mammals. The pattern of the initiation of the ossification evident in the African elephant underscores a possible correlation between the timing of ossification onset and gestation time throughout mammals.

Development of the germinal ridge and ovary in the African elephant (Loxodonta africana).

The follicular reserve and its ontogeny in the elephant are of interest because elephants have the longest reproductive life of all land-based mammals. They also have the longest recorded pregnancy, which allows a protracted view of the series of significant events involved in the development of the embryonic and fetal gonads. The large elephant population of Zimbabwe provided the opportunity to collect conceptuses from elephants culled for management reasons and hunted professionally. Five embryos aged 76-96 days and the ovaries of four fetuses aged 4.8-11.2 months were fixed in 4% buffered formalin and studied by conventional histological sectioning and a stereological protocol to calculate the follicle reserve of each fetus. These observations enabled the conclusion that the migration of primordial germ cells into the indifferent gonad terminates at around 76 days of gestation while entry of oogonia into meiosis along with first follicle formation starts at around 5 months. Peak numbers of follicles are present by mid-gestation towards the end of the 6-month mitotic-meiotic transition period. It appears that the cortex of the elephant fetal ovary at mid-gestation (11 months) has already reached a developmental stage exhibited by the ovaries of many other mammals at full term.

Growth and development of the ovary and small follicle pool from mid fetal life to pre-puberty in the African elephant (Loxodonta africana).

BACKGROUND: Follicle numbers and developing ovarian morphology, particularly with reference to the presence of interstitial tissue, are intimately linked within the ovary of the African elephant during the period spanning mid-gestation to puberty. These have not been previously quantified in any studies. The collection of 7 sets of elephant fetal ovaries between 11.2 and 20.2 months of gestation, and 29 pairs of prepubertal calf ovaries between 2 months and 9 years of age during routine management off-takes of complete family groups in private conservancies in Zimbabwe provided an opportunity for a detailed study of this period. RESULTS: The changing morphology of the ovary is described as the presumptive cortex and medulla components of the fetal ovary settled into their adult form. Interstitial tissue dominated the ovary in late fetal life and these cells stained strongly for 3ß-hydroxysteroid dehydrogenase. This staining continued postnatally through to 4.5 years of age suggesting continued secretion of progestagens by the ovary during this period. The considerable growth of antral follicles peaked at 28% of ovarian volume at around 16.7 months of fetal age. The numbers of small follicles (primordial, early primary and true primary), counted in the cortex using stereological protocols, revealed fewer small follicles in the ovaries of animals aged 0 to 4.5 years of age than during either late fetal life or prepubertal life. CONCLUSIONS: The small follicle populations of the late-fetal and prepubertal ovaries of the African elephant were described along with the changing morphology of these organs. The changes noted represent a series of events that have been recorded only in the elephant and the giraffe species to date. The expansion of the interstitial tissue of the fetal ovary and its continued presence in early post natal life may well contribute to the control of follicle development in these early years. Further research is required to determine the reasons behind the variation of numbers of small follicles in the ovaries of prepubertal calves.

Early embryo development in the elephant assessed by serial ultrasound examinations.

The elephant has an extraordinary long pregnancy, lasting 21 months. However, knowledge on embryo development is limited. To date, only single morphological observations of elephant embryo development associated with placentation are available, all lacking correlation to gestational age. The present study describes morphological characteristics of early embryo development in the elephant with exact biometric staging. Six pregnancies in five Asian and one African elephants with known conception dates were followed by 2D and 3D ultrasound, covering the embryonic period from ovulation to day 116 post-ovulation. The embryonic vesicle was earliest observed was on day 50 p.o. The proper embryo was not detected until day 62 p.o. Embryonic heartbeat was first observed on day 71 p.o. The allantois, which became visible as a single sacculation on day 71 p.o. was subdivided in four compartments on day 76 p.o. By day 95 p.o., head, rump, front and hind legs were clearly distinguished. Between days 95 and 103 p.o. the choriovitelline placenta was replaced by the chorioallantoic placenta. A physiological midgut herniation was transiently present between days 95 and 116 p.o. On the basis of the late appearance of the embryonic vesicle, delayed implantation in the elephant is discussed. The study provides a coherent description of elephant embryonic development, formation of the extraembryonic organs and their role in placenta formation, all of which are of interest for both comparative evolutionary studies and the improvement of assisted reproduction techniques.

Foetal age determination and development in elephants.

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.

Detection of fetal gender differences in maternal serum progesterone concentrations of Asian elephants (Elephas maximus).

Previous studies have analyzed total testosterone concentrations in maternal serum for a reliable method of fetal gender determination in Asian elephants (Elephas maximus). The present study investigated the possibility that progesterone concentrations in maternal serum may reflect these testosterone patterns. Weekly serum samples were collected from 17 pregnancies in captive Asian elephants and analyzed via radioimmunoassay (RIA) for progesterone concentrations. Nine and eight cows carried male and female calves, respectively. Mean progesterone concentrations in maternal serum of elephants carrying male calves were greater than in those carrying female calves (P<0.01). Mean progesterone concentrations (based on 5-week means) in maternal serum were greater at weeks 20-55 (P<0.01) and 60-65 (P<0.05) for elephants carrying male calves.

Humans are born too soon: impact on pediatric otolaryngology.

Humans are born 12 months too early. Gestation should be 21 months. Humans evolved to become the pre-eminent animal in the world, but our big brain, bipedalism, and small female pelvic outlet have caused us to pay the price of being born too soon with all of its disadvantages. Early birth has an impact on diseases and disorders encountered by the otolaryngologist, including otitis media, laryngomalacia, tracheomalacia, congenital vocal cord paralysis, subglottic and tracheal stenosis, gastroesophageal reflux, congenital micrognathia, and congenital nasal alar collapse. Many of these conditions improve or resolve completely in the first year of life as an infant's immune system and anatomy matures. Knowledge of this evolutionary process can help us understand why some infants will grow out of certain diseases and disorders encountered in pediatric otolaryngology, while others will not.

Early development of the tush and the tusk of the African elephant (Loxodonta africana).

This early development was studied from a serial histological sections of eight elephant embryos with masses varying between 1 and 240 g. The tush and the tusk develop from one tooth germ in a deciduous to permanent tooth relation. In order to study the mineralization of the dental organ of the tush and cap and bell stage of the tusk, embryos older than 3-months' gestation (weighing more than 250 g) would be required.

Cryopreservation of gametes and embryos of non-domestic species.

Many species of mammals are threatened or endangered. Methods of assisted reproduction that are being used with increasing frequency to produce offspring of domestic animals and humans are often viewed as offering innovative ways to reproduce non-domestic species as well. Uncounted millions of live young of domestic or laboratory species have been produced from gametes and embryos stored at -70 degrees C or below, sometimes for as long as 25 to 35 yrs. Such methods of cryopreservation are now being applied with increasing frequency and urgency to preserve gametes and embryos of non-domestic and threatened species to establish "genome resource banks" or "frozen zoos." But levels of success to produce live young from such cryopreserved gametes or embryos vary considerably from species to species, as well as from individual to individual. It is sometimes thought that differences among species in fundamental characteristics of their gametes may determine the efficacy of cryopreservation and the production of live young. However, it may not be that ineffective cryopreservation is responsible for low success rates. Rather, the limiting factor may be insufficient information and knowledge of the most basic reproductive biology of such non-domestic species. Even standard methods of cryopreservation may be completely adequate to act as a "temporary" expedient to preserve germplasm of non-domestic species to permit time to acquire a fuller understanding of the biology and behavior of non-domestic species.

Sexual differentiation in three unconventional mammals: spotted hyenas, elephants and tammar wallabies.

The present review explores sexual differentiation in three non-conventional species: the spotted hyena, the elephant and the tammar wallaby, selected because of the natural challenges they present for contemporary understanding of sexual differentiation. According to the prevailing view of mammalian sexual differentiation, originally proposed by Alfred Jost, secretion of androgen and anti-Mullerian hormone (AMH) by the fetal testes during critical stages of development accounts for the full range of sexually dimorphic urogenital traits observed at birth. Jost's concept was subsequently expanded to encompass sexual differentiation of the brain and behavior. Although the central focus of this review involves urogenital development, we assume that the novel mechanisms described in this article have potentially significant implications for sexual differentiation of brain and behavior, a transposition with precedent in the history of this field. Contrary to the "specific" requirements of Jost's formulation, female spotted hyenas and elephants initially develop male-type external genitalia prior to gonadal differentiation. In addition, the administration of anti-androgens to pregnant female spotted hyenas does not prevent the formation of a scrotum, pseudoscrotum, penis or penile clitoris in the offspring of treated females, although it is not yet clear whether the creation of masculine genitalia involves other steroids or whether there is a genetic mechanism bypassing a hormonal mediator. Wallabies, where sexual differentiation occurs in the pouch after birth, provide the most conclusive evidence for direct genetic control of sexual dimorphism, with the scrotum developing only in males and the pouch and mammary glands only in females, before differentiation of the gonads. The development of the pouch and mammary gland in females and the scrotum in males is controlled by genes on the X chromosome. In keeping with the "expanded" version of Jost's formulation, secretion of androgens by the fetal testes provides the best current account of a broad array of sex differences in reproductive morphology and endocrinology of the spotted hyena, and androgens are essential for development of the prostate and penis of the wallaby. But the essential circulating androgen in the male wallaby is 5alpha androstanediol, locally converted in target tissues to DHT, while in the pregnant female hyena, androstenedione, secreted by the maternal ovary, is converted by the placenta to testosterone (and estradiol) and transferred to the developing fetus. Testicular testosterone certainly seems to be responsible for the behavioral phenomenon of musth in male elephants. Both spotted hyenas and elephants display matrilineal social organization, and, in both species, female genital morphology requires feminine cooperation for successful copulation. We conclude that not all aspects of sexual differentiation have been delegated to testicular hormones in these mammals. In addition, we suggest that research on urogenital development in these non-traditional species directs attention to processes that may well be operating during the sexual differentiation of morphology and behavior in more common laboratory mammals, albeit in less dramatic fashion.

Species and fetal gender effects on the endocrinology of pregnancy in elephants.

Quantitative and temporal progestin profiles vary during gestation in the elephant, sometimes making it difficult to determine if a pregnancy is progressing normally. The aim of the present study was to determine if circulating progestin variability was related to species or fetal gender effects. A similar comparison also was conducted for secretory profiles of prolactin, relaxin, and cortisol. Overall mean progestin concentrations during gestation in Asian (n = 19) and African (n = 8) elephants were similar; however, the temporal profiles differed (P < 0.001). Concentrations were higher in African elephants during the first half of pregnancy, but then declined to levels below those observed in Asian elephants (P < 0.05). There also was a fetal gender effect in Asian, but not African elephants. Progestin concentrations were higher in Asian cows carrying male calves (n = 9) as compared to those carrying females (n = 10) (P < 0.001). Overall prolactin concentrations were higher in Asian than in African elephants between 8 and 15 months of gestation ( P< 0.001). There were no species differences in the secretory patterns of relaxin. Cortisol was relatively stable until the end of gestation when significant surges were observed, mainly between 8 and 11 days before parturition, and again on the day of birth. In sum, a comparison of progestin patterns between Asian and African elephants identified notable differences related to species and fetal gender. A role for cortisol in the initiation of parturition also was inferred from these data. From a practical standpoint, understanding the factors affecting gestational hormone characteristics and recognizing what the species differences are will help ensure that data used in diagnosing and monitoring elephant pregnancies are properly interpreted.

The developing renal, reproductive, and respiratory systems of the African elephant suggest an aquatic ancestry.

The early embryology of the elephant has never been studied before. We have obtained a rare series of African elephant (Loxodonta africana) embryos and fetuses ranging in weight from 0.04 to 18.5 g, estimated gestational ages 58-166 days (duration of gestation is approximately 660 days). Nephrostomes, a feature of aquatic vertebrates, were found in the mesonephric kidneys at all stages of development whereas they have never been recorded in the mesonephric kidneys of other viviparous mammals. The trunk was well developed even in the earliest fetus. The testes were intra-abdominal, and there was no evidence of a gubernaculum, pampiniform plexus, processus vaginalis, or a scrotum, confirming that the elephant, like the dugong, is one of the few primary testicond mammals. The palaeontological evidence suggests that the elephant's ancestors were aquatic, and recent immunological and molecular evidence shows an extremely close affinity between present-day elephants and the aquatic Sirenia (dugong and manatees). The evidence from our embryological study of the elephant also suggests that it evolved from an aquatic mammal.

Placentation in the African elephant, Loxodonta africana. I. Endocrinological aspects.

Placental and fetal tissues were recovered from the uteri of 59 pregnant elephant that ranged in estimated age from day 18 to month 21 of gestation. Incubation of placenta and fetal gonad, alone or in combination, with tritium-labelled cholesterol, pregnenolone and androstenedione failed to yield any labelled progestagens or oestrogens from placenta, but did produce small amounts of labelled progesterone and 5alpha-dihydroprogesterone from fetal gonad. Immunochemical staining of tissues with four antisera specific for enzymes involved in the steroidogenic pathway revealed no staining in sections of placenta but positive labelling for P450 side chain cleavage enzyme (SCC450) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the interstitial cells that comprise the bulk of the enlarged fetal gonads during the second half of gestation. Saline extracts of placental tissue showed no activity in three different gonadotrophin assays. In view of this endocrinological inactivity in the zonary elephant placenta and the probable reliance on maternal luteal sources of progestagens for maintenance of the pregnant state, the argument is advanced that uncomplicated abortion would probably follow a single administration of a PGF analogue given at any stage of pregnancy. If so, the treatment might constitute an efficacious method for controlling population increases in elephants maintained in enclosed game parks in Africa.