Non-targeted proteomic analysis of Asian elephant (Elephas maximus) seminal plasma using an in-solution digestion technique and liquid chromatography tandem-mass spectrometry.

Seminal plasma proteins have recently been reported to play a significant role as valuable materials for understanding male reproductive biology, identifying causes of fertility problems, and developing reproductive biomarkers. Proteomic analysis of seminal plasma holds promise in advancing the understanding of male Asian elephant reproductive biology. This study aims to explore seminal plasma proteins of Asian elephants and their probable functions to provide fundamental information about male reproduction in this species. The protein solution from pooled seminal plasma from 10 bulls (a total of 33 ejaculates) was digested into peptides and identified using LC-MS/MS. Out of 986 proteins, 597 were mapped and matched with 58 species in UniProt databases, including Elephas maximus. These mapped proteins were mostly involved in binding function, catalytic activity, cellular process, and metabolic process. Only 29 mapped proteins were recognized to be related in reproductive process, mainly associated in spermatogenesis and sperm capacitation. Additionally, several seminal plasma proteins related to fertility or semen quality in other mammals were also found in Asian elephant semen, such as keratin type I, aldose reductase, thrombospondon-1, fibronectin 1, platelet-activating factor acetyl hydrolase, mannosidase, and semenogelin-2. This discovery clearly reveals the beneficial protein profile in seminal plasma of the Asian elephant and serves as a crucial step in investigating infertility and poor semen quality in this valuable species.

Relationship among Serum Progestagens, Cortisol, and Prolactin in Pregnant and Cycling Asian Elephants in Thailand.

The aim of this study was to examine relationships among serum progestagens, cortisol, and prolactin in pregnant and normal cycling Asian elephants living in tourist camps in northern Thailand. Samples were collected twice a month for 22 months from nine elephants. Of those, four were pregnant (24.3 ± 2.9 years of age; range 21−28 years) and five (20.2 ± 9.6 years; range 8−34 years) exhibited normal ovarian cycles based on serum progestagen analyses. Gestation was divided into three periods: 1st (week 1−31), 2nd (week 32−62), and 3rd (week 63 to parturition), while the estrous cycle was divided into the follicular and luteal phases. Serum progestagens were higher during the luteal phase of the cycle (p < 0.003), whereas cortisol and prolactin were similar. In pregnant elephants, there were no differences in serum progestagens or cortisol concentrations across the three gestational periods, whereas prolactin concentrations increased significantly during the 2nd and 3rd periods (p < 0.0001). By contrast, prolactin concentrations in nonpregnant elephants were consistently low throughout the ovarian cycle. In one cycling female, prolactin concentrations were similar to pregnant elephants, perhaps because she was an allomother to two calves. Another cycling female exhibited consistently elevated cortisol concentrations, 5 to 10 times higher than the other elephants. There were no correlations between serum progestagens, cortisol, and prolactin throughout gestation; however, serum progestagens and cortisol were positively related in cycling elephants (r = 0.386, p < 0.001). From our results, there were a number of individual differences in reproductive hormonal patterns, so it is important to develop personalized monitoring programs for each elephant to enhance breeding success and create sustaining captive populations of elephants in Asia.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls.

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 µg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

Potential of a gonadotropin-releasing hormone vaccine to suppress musth in captive male Asian elephants (Elephas maximus).

Musth in adult bull elephants is a period of increased androgen concentrations ranging from a few weeks to several months. For captive elephant bull management, musth presents a serious challenge because of the aggressive behavior of musth bulls toward people and other elephants. Commercially available GnRH vaccines have been shown to suppress testicular function by interrupting the hypothalamo-pituitary-gonadal (HPG) axis in many species. The aim of this study was to test the efficacy of a GnRH vaccine in elephant bulls for suppressing the HPG axis and mitigating musth-related aggressive behavior. Five adult Asian elephant bulls (22-55 years old) were immunized with a GnRH vaccine starting with an initial injection 2-4 months before the predicted musth period, and followed by three boosters at approximately 4-week intervals. Blood samples were collected twice weekly for hormone and antibody titer analysis. An increase in GnRH antibody titers was observed in all bulls after the second or third booster, and titers remained elevated for 2-3 months after the final booster. Musth was attenuated and shortened in three bulls and postponed completely in two. We conclude that GnRH vaccination is capable of suppressing symptoms of musth in adult bull elephants. With appropriate timing, GnRH vaccination could be used to control or manage musth and aggressive behavior in captive elephant bulls. However, more work is needed to identify an optimal dose, booster interval, and vaccination schedule for complete suppression of testicular steroidogenesis.

The in vitro quality of frozen-thawed Asian elephant (Elephas maximus) spermatozoa in semen supplemented with Equex STM paste and oxytocin during and after cryopreservation.

The effects of Equex STM paste (Equex) and oxytocin (OT) on the in vitro quality of frozen-thawed Asian elephant sperm were investigated in the study. The viability of frozen-thawed sperm was significantly higher in the Equex-treated (1 and 2%) groups than in the control group. There were no differences in the examined sperm parameters among the control and OT-treated (0.05-5IU) groups.

Mycobacterium tuberculosis infection of domesticated Asian elephants, Thailand.

Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S-23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.

Successful artificial insemination in the Asian elephant (Elephas maximus) using chilled and frozen-thawed semen.

BACKGROUND: Artificial insemination (AI) using frozen-thawed semen is well established and routinely used for breeding in various mammalian species. However, there is no report of the birth of elephant calves following AI with frozen-thawed semen. The objective of the present study was to investigate the fertilizing ability of chilled and frozen-thawed semen in the Asian elephant following artificial insemination (AI). METHODS: Semen samples were collected by from 8 bulls (age range, 12-to 42-years) by manual stimulation. Semen with high quality were either cooled to 4 degrees C or frozen in liquid nitrogen (-196 degrees C) before being used for AI. Blood samples collected from ten elephant females (age range, 12-to 52-years) were assessed for estrus cycle and elephants with normal cycling were used for AI. Artificial insemination series were conducted during 2003 to 2008; 55 and 2 AI trials were conducted using frozen-thawed and chilled semen, respectively. Pregnancy was detected using transrectal ultrasonography and serum progestagen measurement. RESULTS: One female (Khod) inseminated with chilled semen became pregnant and gave birth in 2007. The gestation length was 663 days and the sex of the elephant calf was male. One female (Sao) inseminated with frozen-thawed semen showed signs of pregnancy by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography. CONCLUSION: This is the first report showing pregnancy following AI with frozen-thawed semen in the Asian elephant. Successful AI in the Asian elephant using either chilled or frozen-thawed semen is a stepping stone towards applying this technology for genetic improvement of the elephant population.

Induction of the ovulatory LH surge in Asian elephants (Elephas maximus): a novel aid in captive breeding management of an endangered species.

A unique feature of the reproductive physiology of Asian elephants (Elephas maximus) is the occurrence of two LH surges before ovulation, instead of one. An anovulatory LH (anLH) surge, the function of which is unknown, occurs consistently 3 weeks before the ovulatory LH (ovLH) surge that induces ovulation. Thus, the ability to induce an ovLH surge would be useful for scheduling natural mating or artificial insemination. The present study tested the efficacy of a gonadotrophin-releasing hormone agonist (GnRH-Ag) to induce LH surges during the follicular phase of the oestrous cycle, which resulted in varied LH responses, but generally none were as high as previously documented natural surges. Thus, for the ovulation-induction trials, nine females were administered 80 microg GnRH-Ag intravenously at three time periods during the oestrous cycle, namely the anovulatory follicular phase, the ovulatory follicular phase and the luteal phase. During the late anovulatory follicular phase, nine of 10 females (90%) responded with an immediate LH surge followed 15-22 days later by an ovLH surge or a post-ovulatory increase in progestagens. In contrast, despite responding to the GnRH-Ag with an immediate increase in LH, none of the females treated during other periods of the oestrous cycle exhibited subsequent ovLH surges. One cow got pregnant from natural mating following the induced ovLH surge. In conclusion, ovLH induction is possible using a GnRH-Ag, but only during a specific time of the anovulatory follicular phase.

Use of genital inspection and female urine tests to detect oestrus in captive Asian elephants.

Captive Asian elephant (Elephas maximus) populations are decreasing due to low birth rates compared to wild elephants. Improving oestrous detection in female elephants is required to ensure successful mating in captive and semi-captive herds. Responsive behaviours of eight semi-captive bull elephants to the uro-genital area (genital inspection test) or urinary pheromones (urine test) of 14 female elephants throughout the oestrous cycle were evaluated. Weekly blood samples were collected for 27 consecutive months (14 months for the genital inspection test and 13 months for the urine test) from female elephants to characterize the patterns of circulating progestagen. Responsive behaviours of bulls were compared between females in the follicular versus the luteal phase of the cycle. The sensitivity and specificity of the genital inspection test were 65% and 68%, while those of the urine test were 52% and 61%, respectively. The bulls showed significantly higher "genital inspection", "flehmen from genital area" and "trunk on back" behaviours during the genital inspection test, and "flehmen" behaviours during the urine test in oestrous than in non-oestrous females. In sum, this study showed that monitoring sexual behaviours of Asian elephant bulls towards females or their urine can be used to detect the oestrous period. Although the sensitivity and specificity of both tests were not as high as expected, still, these methods appear to be more efficient at detecting oestrous than traditional methods based on mahout estimations of female receptivity. The use of genital inspection and urine tests may lead to more successful matings and thus to creating self-sustaining populations of captive elephants in range countries.

Potential factors affecting semen quality in the Asian elephant (Elephas maximus).

BACKGROUND: One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant METHODS: Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. RESULTS: The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05). CONCLUSION: This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species.

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant.

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.