Imke Lueders

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.

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.

Role of the Double Luteinizing Hormone Peak, Luteinizing Follicles, and the Secretion of Inhibin for Dominant Follicle Selection in Asian Elephants (Elephas maximus)

Elephants express two luteinizing hormone (LH) peaks timed 3 wk apart during the follicular phase. This is in marked contrast with the classic mammalian estrous cycle model with its single, ovulation-inducing LH peak. It is not clear why ovulation and a rise in progesterone only occur after the second LH peak in elephants. However, by combining ovarian ultrasound and hormone measurements in five Asian elephants (Elephas maximus), we have found a novel strategy for dominant follicle selection and luteal tissue accumulation. Two distinct waves of follicles develop during the follicular phase, each of which is terminated by an LH peak. At the first (anovulatory) LH surge, the largest follicles measure between 10 and 19.0 mm. At 7 ± 2.4 days before the second (ovulatory) LH surge, luteinization of these large follicles occurs. Simultaneously with luteinized follicle (LUF) formation, immunoreactive (ir) inhibin concentrations rise and stay elevated for 41.8 ± 5.8 days after ovulation and the subsequent rise in progesterone. We have found a significant relationship between LUF diameter and serum ir-inhibin level (r2 = 0.82, P < 0.001). The results indicate that circulating ir-inhibin concentrations are derived from the luteinized granulosa cells of LUFs. Therefore, it appears that the development of LUFs is a precondition for inhibin secretion, which in turn impacts the selection of the ovulatory follicle. Only now, a single dominant follicle may deviate from the second follicular wave and ovulate after the second LH peak. Thus, elephants have evolved a different strategy for corpus luteum formation and selection of the ovulatory follicle as compared with other mammals.

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.

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.

SUCCESSFUL NONSURGICAL ARTIFICIAL INSEMINATION AND HORMONAL MONITORING IN AN ASIATIC GOLDEN CAT (CATOPUMA TEMMINCKI)

Abstract: Since it is reported to be difficult to establish Asiatic golden cat (Catopuma temmincki) breeding pairs in captivity as a result of overaggressive behavior of the male, artificial insemination (AI) may be a desired option by which to achieve pregnancy. This approach was chosen in the present case involving a nulliparous, 6-yr-old female cat that was inseminated transcervically during a naturally occurring estrus, which therefore required only a single general anesthetic procedure. On day 4 of estrus behavior, the male was anesthetized and semen was collected via urethral catheterization (UC) to recover spermatozoa in high concentration followed by electro-ejaculation (EE) to obtain additional semen and seminal fluid. The fresh UC semen, totaling 180 μl in volume and containing spermatozoa showing 55–70% sperm motility, was inseminated 2.5 hr later via a commercial cat urinary catheter passed through the cervix into the uterus. Immediately afterwards, the EE fraction (100 μl) was inseminated deeply into the dorsal medial fold of the vagina. The GnRH analogue Receptal® (0.75 ml, i.m.) was given during anesthesia in an attempt to induce ovulation. Increasing fecal concentrations of progesterone after AI and a significant rise in fecal prostaglandin F2α metabolite (PGFM) concentrations (P < 0.0001) from day 45 post-AI indicated that the cat had conceived, and it produced healthy twin cubs after an 84-day gestation.

SUPRESSION OF TESTICULAR FUNCTION IN A MALE ASIAN ELEPHANT (ELEPHAS MAXIMUS) TREATED WITH GONADOTROPIN-RELEASING HORMONE VACCINES

Abstract:  The ability to control testosterone concentrations and sperm production is of great interest in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. GnRH vaccination may pose an alternative to surgical castration. This is a case report of a male Asian elephant treated with two commercial GnRH vaccines (Equity® and Improvac®). Beginning at the age of 7 yr, the male was vaccinated monthly for 6 consecutive months, then every 6 mo and, finally, every 12 to 24 mo over a period of 6 yr. In order to evaluate the GnRH vaccine as a potential method of immunologic castration, behavioral observations, testosterone level analysis, body weights, ultrasound examinations, and semen collection were part of the routine monitoring of this bull (no. 1) and a half-brother (bull 2) who remained untreated and served as control. The results showed a decrease in serum testosterone concentrations after the second booster. Levels stayed continuously below 5.0 ng/ml within the study period. The combined testicle diameter of 9.03 ± 0.3 cm prior to treatment had decreased to a size of 6.93 ± 0.19 cm (P < 0.001) when measured 2 yr later. Accessory sex gland fluid content disappeared and penile atrophy was observed. Semen collections yielded no spermatozoa 1 yr after the initial treatment. Bull 1 showed slowed weight gain as compared to bull 2 and, due to its friendly temperament and the absence of musth, remained in free contact. This report documents the GnRH vaccine as a possible noninvasive and inexpensive method for immunecastration.

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.

Tracheal Resection in a Secretary Bird (Sagittarius serpentarius) with Granulomatous, Foreign-body Induced Tracheitis

Abstract A 24-year-old female secretary bird (Sagittarius serpentarius) was presented with acute, mild dyspnea occurring only during feeding times. Despite initial conservative therapy consisting of antibiotics and antifungal, antiparasitic, and anti-inflammatory drugs, the dyspnea worsened progressively, resulting in severe respiratory distress. Radiographs of the trachea suggested stenosis in the caudal one-third of the trachea. Tracheal endoscopy revealed an obstruction of approximately 90% of the tracheal lumen, in addition to mild suspected aspergillosis of the air sacs. Tracheal resection and anastomosis were performed, during which 1.5 cm of abnormal trachea was removed. Histopathologic examination showed severe granulomatous tracheitis, most likely induced by foreign body material. Respiratory signs resolved immediately postoperatively. Antibiotic and anti-inflammatory therapy continued for another 7 days and the bird was treated with antifungals for a total of 45 days. The bird recovered uneventfully. We encourage tracheal resection and anastomosis for severe tracheal stenosis even in aged, large birds of prey that are managed in large aviaries.