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 w10 days after the respective LH peak. After ovulation, it took 29.8G5.0 days for the acCLs to reach their maximum diameter, whereas the ovCL reached a significantly larger size (33.2G2.3 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.

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)(1999)(2000)(2001)(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.

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.

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.

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