Charlie Gray

Gestating for 22 months: luteal development and pregnancy maintenance in elephants.

The corpus luteum, a temporally established endocrine gland, formed on the ovary from remaining cells of the ovulated follicle, plays a key role in maintaining the early mammalian pregnancy by secreting progesterone. Despite being a monovular species, 2–12 corpora lutea (CLs) were found on the elephant ovaries during their long pregnancy lasting on average 640 days. However, the function and the formation of the additional CLs and their meaning remain unexplained. Here, we show from the example of the elephant, the close relationship between the maternally determined luteal phase length, the formation of multiple luteal structures and their progestagen secretion, the timespan of early embryonic development until implantation and maternal recognition. Through three-dimensional and Colour Flow ultrasonography of the ovaries and the uterus, we conclude that pregnant elephants maintain active CL throughout gestation that appear as main source of progestagens. Two LH peaks during the follicular phase ensure the development of a set of 5.4 ± 2.7 CLs. Accessory CLs (acCLs) form prior to ovulation after the first luteinizing hormone (LH) peak, while the ovulatory CL (ovCL) forms after the second LH peak. After five to six weeks (the normal luteal phase lifespan), all existing CLs begin to regress. However, they resume growing as soon as an embryo becomes ultrasonographically apparent on day 49 ± 2. After this time, all pregnancy CLs grow significantly larger than in a non-conceptive luteal phase and are maintained until after parturition. The long luteal phase is congruent with a slow early embryonic development and luteal rescue only starts ‘last minute’, with presumed implantation of the embryo. Our findings demonstrate a highly successful reproductive solution, different from currently described mammalian models.

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.

Luteogenesis during the estrous cycle in Asian elephants (Elephas maximus)

The occurrence of multiple corpora lutea (CLs) in the ovaries of the cycling and pregnant elephant, a monovulatory mammal, has driven scientific discussions during the past five decades. However, fundamental knowledge on luteogenesis is lacking. In this long-term study, CL formation and regression throughout the estrous cycle were monitored using transrectal 2D- and 3D ultrasonography in 33 captive Asian elephants. Serum or urinary progestagens (P(m)) were measured to determine the reproductive cycle stage. In seven females, serum P(m) and LH concentrations were directly related to ovarian events. We have found two different modalities of luteal development: one for the accessory CL (acCL) and one for the ovulatory CL (ovCL). acCLs were derived from luteinization of larger, subordinate follicles after the first anovulatory LH peak. The dominant follicle produced the largest CL after the second (ovulatory) LH peak. The first luteal tissue formation became visible ∼ 10 days after the respective LH peak. After ovulation, it took 29.8 ± 5.0 days for the acCLs to reach their maximum diameter, whereas the ovCL reached a significantly larger size (33.2 ± 2.3 mm, P<0.0001) about 10-15 days later. All CLs were visible throughout the new follicular phase, with some of the larger ones still present in the subsequent luteal period. In this study, we have demonstrated that Asian elephants have evolved a novel method for luteal development and function, and by repeatedly forming two types of distinctly different CLs for every reproductive cycle, they have ensured that there will be sufficient luteal capacity for maintaining a 22-month pregnancy should conception occur.

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.

Ovarian Ultrasonography Correlated with Fecal Progestins and Estradiol During the Estrous Cycle and Early Pregnancy in Giraffes (Giraffa camelopardalis rothschildi)

Fecal and urinary progestin analyses have shown that giraffes express a short reproductive cycle, averaging 15 days, compared with other large ruminants. However, actual ovarian events have not been correlated with the hormonal pattern. In this study, mature cycling female Rothschild giraffes (Giraffa camelopardalis rothschildi) were repeatedly examined by transrectal ultrasonography to correlate ovarian function with changes in fecal progestin (fP4 [nc = 6]) and estradiol (fE2 [nc = 6]) and serum progestin (nc = 2) as measured by enzyme immunoassay. Five females became pregnant and were monitored during early gestation. In this study, we discovered that hormone values for fP4 in cycling giraffes do not correlate with the classic profile of follicular development, ovulation, and luteogenesis. The corpus luteum (CL) and the next dominant follicle were forming simultaneously. A mean ± SD peak in fE2 of 254.92 ± 194.76 ng/g and subsequent ovulation occurred as early as 1 day after the fall in fP4. In pregnant giraffes, the CL reached a diameter significantly larger (mean ± SD, 41.02 ± 2.70 mm; P = 0.0126) than that during the cycle (33.48 ± 2.80 mm), while follicular activity and fluctuating fE2 were still present. With this research, we demonstrated that the progesterone profile typically used to characterize the ovarian cycle does not correlate with luteal development in the ovaries of this species. Furthermore, we conclude that the giraffe could have evolved a short reproductive cycle because of the almost parallel order of ovarian events.

Estrus induction in white rhinoceros (Ceratotherium simum)

The estrous cycle length in the white rhinoceros (Ceratotherium simum) is either 4 or 10 wk. The cause(s) for this variation as well as the poor fertility rate in captivity remains under debate in this species. Most captive adult white rhinoceros undergo long anovulatory periods without luteal activity which are considered a major reason for their low reproductive rate. In this study, the synthetic progestin chlormadinone acetate (CMA) was tested in combination with hCG or the GnRH analogue deslorelin for its efficiency to induce ovulation in fourteen females without luteal activity and in three, regular cycling females. HCG (N = 12), injectable GnRH analogue (N = 8) and GnRH analogue implants (N = 15) were given to induce ovulation after CMA treatment. Treatment success was determined using both transrectal ultrasonography and progesterone metabolite EIA analysis. A preovulatory sized follicle (3.5 ± 0.1 cm) or a corpus luteum (5.1 ± 0.7) was present on the ovary one day after induction in 93.1% of the treatments. Despite this high rate of ovarian response, ovulation rate differed between the study groups. The ovulation rate for hCG, injectable GnRH analogue and GnRH analogue implants was 66.7%, 62.5% and 93.3%, respectively. Ovulation rate in cyclic females treated with GnRH implants was 100% (6/6) compared with 89% (8/9) in females without luteal activity receiving the same treatment. The length of the estrous cycle when induced with hCG was 4 wk (85.7%). The estrous cycle when induced with GnRH analogue was predominantly 10 wk long. Two females without luteal activity treated with GnRH became pregnant. In conclusion, CMA in combination with GnRH analogue implants was highly effective to induce ovulation in white rhinoceroses and thus can contribute to efforts aimed at increasing natural mating and reproductive rates in the captive white rhinoceros population.

Sonomorphology of the reproductive tract in male and pregnant and non-pregnant female Rothschild's giraffes (Giraffa camelopardalis rotschildi)

The application of real-time-B-mode ultrasonography to wild and zoo animal medicine has been shown to improve the understanding of reproductive physiology in many species. Ultrasound technology is especially helpful for monitoring urogenital health, which in turn has advantages for giraffe breeding and welfare in captivity. This study aimed to ultrasonographically describe the genital organs of reproductively healthy male and female giraffes. Through the use of a restrainer, repeated rectal ultrasound examinations were performed over a 2 year period in 2.6 Rothschilds giraffes. Changes in ovarian activity were monitored throughout four different reproductive stages in the females and included immature, mature-cycling, pregnancy, post-partum-period. In the immature giraffes the ovaries showed multiple follicles of which larger ones luteinized to form pseudo-corpora lutea. By comparison, in the mature giraffes the dominant follicle reached an ovulatory diameter of 18.5+/-0.89 mm. After ovulation, a single corpus luteum rapidly formed and reached a maximum diameter of 33.0+/-2.4mm on average. Pregnancy was detected for the first time by the embryonic vesicle, visualized around 28 days post copulation. Follicular development remained ongoing during early pregnancy. In the males, as in other ruminants, the bulbourethral glands and the seminal vesicles were prominent, whereas the prostate gland was indistinct. Knowledge about the reproductive tract morphology and physiology is necessary for diagnosing medical disorders and abnormalities in giraffes. The aim of this study was to help consolidate the current knowledge on basic reproductive parameters for this species.

Ultrasonographically documented early pregnancy loss in an Asian elephant (Elephas maximus).

Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.

Nonsurgical repair of an umbilical hernia in two Asian elephant calves (Elephas maximus).

Abstract Umbilical hernias were diagnosed in two captive-born, female Asian elephant (Elephas maximus) calves several weeks after birth. Daily manual reduction of the hernias for 5 wk in the first case and for 5 mo in the second resulted in complete closure of the defects. Nonsurgical repair of uncomplicated, fully reducible umbilical hernias in Asian elephants can be an alternative to surgery.

A simple, field-friendly technique for cryopreserving semen from Asian elephants (Elephas maximus)

The specific objectives of the present study were to investigate the effects of manual seeding, differing freeze and thaw rates as well as storage for 24h at 4°C prior to cryopreservation on post-thaw sperm quality in Asian elephants. Extended semen was cooled in an equitainer to 4°C, frozen in liquid nitrogen vapour at various rates with and without manual seeding or in a dry shipper and thawed at 37, 50 and 75°C. There was a significant effect of freeze rate on post-thaw motility (P<0.0001) and acrosomal integrity (P<0.005). The faster freeze rates in the dry shipper and at 1cm or 2cm above liquid nitrogen consistently provided better cryopreservation than slower freezing rates. Thaw temperature had no effect on post-thaw semen quality but there was an interaction between freeze and thaw rates with higher thaw rates resulting in superior post-thaw semen quality in straws frozen at fast rates. Storage of samples prior to freezing had a detrimental effect on post-thaw semen quality. In summary, our results indicate cooling extended semen in an equitainer and cryopreserving it by placing straws directly in a dry shipper is a simple technique for effectively cryopreserving Asian elephant semen in the field or zoo.