Robert Hermes

Ultrasonography of the Urogenital Tract in Elephants (Loxodonta africana and Elephas maximus): An Important Tool for Assessing Male Reproductive Function

The success rate of captive elephant breeding programs worldwide is poor. Along with undiagnosed reproductive disorders in females and fatal diseases such as the newly discovered herpesvirus infection, male infertility now is considered a major contributing factor in the failure to maintain self-sustaining captive populations. To address questions related to male reproductive dysfunction, approximately 309 ultrasonographic assessments combined with semen collection were performed in captive (n = 10) and wild (n = 4) African (Loxodonta africana) and captive (n = 61) Asian (Elephas maximus) elephants. Bulls ranged from 4 to 50 years of age and were examined at 9 institutions in North America, 13 in Europe, 2 in Africa, and 7 in Asia. About half of the reproductive assessments were performed in protected contact situations with elephants handled in a restraint device, and half involved assessments of trained Asian bulls managed in free contact. Four wild African and two Asian elephant bulls were evaluated after receiving general anesthesia. Transrectal ultrasound was used to characterize the morphology and functionality of the entire urogenital tract, including the testes and accessory sex organs. Bulls were categorized on the basis of breeding status (breeders vs. non-breeders) and social history (i.e., type of interaction with conspecifics and keepers). Most of the bulls were non-breeders (designated Types I–V). Type I (n = 3 African, 6 Asian) and Type V (n = 1 Asian) were immature and castrate, respectively. On the basis of keeper evaluations, Type II bulls (n = 2, 4) were subordinate to older cows and keepers, whereas Type III bulls (n = 4, 28) were dominated by other bulls. Type IV (n = 1, 8) were older bulls of unknown history that exhibited numerous testicular pathologies resulting in poor semen quality. Non-breeding bulls included those that were exposed to females, but failed to breed, as well as those that had no opportunities to breed. Type VI individuals (n = 4, 14) were proven breeders. The percentage of observable reproductive tract pathology in adult males was remarkably low (14%), even in older bulls. However, apparent infertility of non-organic cause (i.e., not due to specific anatomical abnormalities) in these otherwise healthy bulls was high (32%). Semen quality varied markedly in ejaculates collected from the same bull, as well as from different bulls. In conclusion, although many of these bulls could serve as semen donors for natural mating or artificial insemination, the inconsistent production of good-quality ejaculates raises questions as to the reliability of these individuals to participate in breeding programs. The apparent inhibitory effect of suppressive social interactions on reproductive potential also needs to be investigated. Ultrasound examinations combined with semen collection should be conducted periodically to estimate the reproductive value of each bull and determine whether altered management strategies are needed to enhance captive breeding. Zoo Biol 19:333–345, 2000.

Ultrasonography as an important tool for the development and application of reproductive technologies in non-domestic species.

Ultrasound imaging in reproductive sciences offers new opportunities regarding optimization of the induction of the sexual cycle and ovulation, superovulation regimes, contraception programs, semen collection and testicular sperm extraction techniques, ovum pick up and ovarian transplantation procedures, as well as the application of artificial insemination, embryo collection and transfer. In non-domestic species, most of which lack basic data, ultrasonography is an ideal tool to study reproductive biology in both captive and wild populations. The use of this imaging modality led us to develop new, or modify established, reproductive technologies. Ultrasonography has been an integral part of over 200 assisted reproduction procedures in 17 mammalian species performed by our research team between 1992 and 1999. These procedures included the initial characterization of sexual cycles, hormonal cycle induction, semen collection by electroejaculation or manual stimulation, non-surgical artificial insemination (AI), non-surgical embryo transfer and temporary hormonal contraception. For these investigations, a variety of newly developed equipment was applied and species-specific hormonal treatments designed. We used several commercial and customized ultrasound systems with a variety of technical features. Some relevant improvements of these applications will be described and the role of ultrasonography elucidated to.

First successful artificial insemination with frozen-thawed semen in rhinoceros

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of approximately 135 x 10(6) motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of approximately 500 x 10(6) motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. 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 between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. 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 managing genetic diversity in these endangered mammals.

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa.

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen-thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2+/-5.4%, motility following 30 min incubation at 37 degrees C: 58.5+/-6.0% and following 3h incubation: 21.7+/-7.6%, intact acrosome: 57.1+/-5.2%, normal morphology: 52.0+/-5.8% and viability: 67.3+/-6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.

Ultrasonography of the estrous cycle in female African elephants (Loxodonta africana)

The endocrinology of the elephant estrous cycle has been well characterized, but little emphasis has been placed on evaluating corresponding changes in the reproductive tract. Ultrasound was used to document changes in reproductive tract morphology throughout the estrous cycle in four cycling female African elephants. During a 7-month period, frequent ultrasound examinations (n = 190) during the luteal and non-luteal phase were compared with serum progesterone and luteinizing hormone (LH) concentrations during a 7-month period. Ultrasonographic images documented vaginal and cervical edema and changes in mucus consistency during the non-luteal phase. The cross-sectional diameter of the endometrium showed a dramatic increase during the non-luteal phase and followed cyclic changes. A different pattern of follicular development on the ovary was associated with the two LH surges. Follicle growth associated with the first, anovulatory LH surge was characterized by the formation of multiple small follicles, in contrast to the maturation of a single large follicle at the second, ovulatory LH (ovLH) surge. Ovulation and the subsequent formation of a corpus luteum (CL) were observed only after the ovLH surge. Ultrasound data in combination with endocrine assessments suggest that the African elephant is non-ovulatory, although multiple non-ovulatory luteal structures developed during the late nonluteal phase of each cycle. Both ovulatory CL and non-ovulatory luteal structures were present only through one cycle and regressed at the end of the luteal phase in conjunction with the drop in serum progesterone. We conclude that

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni)

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p<0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p<0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p<0.01, p<0.03, p<0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p<0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.

Skewed birth sex ratio and premature mortality in elephants

Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n=487) and premature deaths (PD, <5 years of age; n=164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n=3070; PD, n=738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P=0.044) and in births following artificial insemination (0.83, P=0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21-23%, it was almost double (40-45%) in all other captive populations. In zoo populations, 68-91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P=0.024 and 0.56, P<0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.

Obstetrics in elephants

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

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.

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n=9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.