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

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Methods</p> <p>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°C or frozen in liquid nitrogen (-196°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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Infectious Diseases Management in Asian Elephant : TB and EEHV-HD

Asian elephant’s health, well-being and population sustainability are affected by several life-threatening diseases. Tuberculosis (TB), with potential zoonotic impact, and EEHV-HD, live threatening especially for young elephants, are amongst the most serious ones. Besides, they have the potential to severely affect the economy, particularly in relation to tourism. Infections of elephants with M. tuberculosis and EEHV have been reported worldwide, in Thailand frequent contact between elephants and M. tuberculosis infected human individuals, as well as between wild and captive elephants is likely. Our study, three reacted positive in a serological test (TB STAT Pak, Chembio). In serum of one of these, antibodies were found retrospectively, 23 months prior to bacterial culture from trunk wash samples. Sequence analysis of M. tuberculosis isolates from the four animals classified them as ancient strains (n=1) based on presence of a M. tuberculosis specific deletion (TbD1) and modern strains (n=3) identical to M. tuberculosis ATCC 2794. Detection of interferon gamma (IFN-γ) in a range of cytokines produced upon stimulation with M. tuberculosis derived antigens, is used for diagnosis at the early stage of infection with M. tb. Detection levels ranging between 1 and 10,000 pg/ml and able to detect native elephant IFN-γ of both African and Asian elephant whole blood cultures. Among Asian elephants, of different TB status, detection of IFN-γ upon in vitro stimulation of whole blood samples with ESAT6/CFP10, PPDB and PPDA, was negative for samples from non-infected elephants, as well as those from an elephant suspected of TB. Whole blood cell stimulation of a M. tb infected elephant with antigens ESAT6/CFP10 PPDB and PPDA resulted in detection of interferon gamma in all cultures, though to a lesser extent when stimulated with PPDA as a control for non MTBC environmental mycobacteria contact. ELISA results of serum samples of 708 elephants were subjected to Latent Class Analysis (LCA) to predict their serological TB statuses. Depending on LCA approaches, in case population versus individual elephant based, relative numbers (percentages) for the different statuses were “positive” 15-17.3%, “inconclusive” 48.7-50% and “negative” 34-35% of the animals. In this study, the North region had the lowest percentages of elephants predicted to be of positive serological TB status. The West region and to a lesser extend the other regions showed clearly higher percentages. Haemorrhagic Disease caused by Elephant Endotheliotropic Herpes Virus (EEHV-HD) we performed a cross sectional serological survey, using an EEHV1 glycoprotein B (gB) specific antibody ELISA. Of 994 elephants included in the study 42.3% were positive and 57.7%negative, while none were inconclusive, based on the criterion that a serum sample is considered positive if at least one serum dilution tested positive. Associations between seropositivity and potential risk factors for EEHV infection in the final multivariable regression model the variable “region” was represented. Finally, publication of this thesis will increase professional and public awareness of the threats of TB and EEHV infection in Elephants in Thailand

Infectious Diseases Management in Asian Elephant : TB and EEHV-HD

Asian elephant’s health, well-being and population sustainability are affected by several life-threatening diseases. Tuberculosis (TB), with potential zoonotic impact, and EEHV-HD, live threatening especially for young elephants, are amongst the most serious ones. Besides, they have the potential to severely affect the economy, particularly in relation to tourism. Infections of elephants with M. tuberculosis and EEHV have been reported worldwide, in Thailand frequent contact between elephants and M. tuberculosis infected human individuals, as well as between wild and captive elephants is likely. Our study, three reacted positive in a serological test (TB STAT Pak, Chembio). In serum of one of these, antibodies were found retrospectively, 23 months prior to bacterial culture from trunk wash samples. Sequence analysis of M. tuberculosis isolates from the four animals classified them as ancient strains (n=1) based on presence of a M. tuberculosis specific deletion (TbD1) and modern strains (n=3) identical to M. tuberculosis ATCC 2794. Detection of interferon gamma (IFN-γ) in a range of cytokines produced upon stimulation with M. tuberculosis derived antigens, is used for diagnosis at the early stage of infection with M. tb. Detection levels ranging between 1 and 10,000 pg/ml and able to detect native elephant IFN-γ of both African and Asian elephant whole blood cultures. Among Asian elephants, of different TB status, detection of IFN-γ upon in vitro stimulation of whole blood samples with ESAT6/CFP10, PPDB and PPDA, was negative for samples from non-infected elephants, as well as those from an elephant suspected of TB. Whole blood cell stimulation of a M. tb infected elephant with antigens ESAT6/CFP10 PPDB and PPDA resulted in detection of interferon gamma in all cultures, though to a lesser extent when stimulated with PPDA as a control for non MTBC environmental mycobacteria contact. ELISA results of serum samples of 708 elephants were subjected to Latent Class Analysis (LCA) to predict their serological TB statuses. Depending on LCA approaches, in case population versus individual elephant based, relative numbers (percentages) for the different statuses were “positive” 15-17.3%, “inconclusive” 48.7-50% and “negative” 34-35% of the animals. In this study, the North region had the lowest percentages of elephants predicted to be of positive serological TB status. The West region and to a lesser extend the other regions showed clearly higher percentages. Haemorrhagic Disease caused by Elephant Endotheliotropic Herpes Virus (EEHV-HD) we performed a cross sectional serological survey, using an EEHV1 glycoprotein B (gB) specific antibody ELISA. Of 994 elephants included in the study 42.3% were positive and 57.7%negative, while none were inconclusive, based on the criterion that a serum sample is considered positive if at least one serum dilution tested positive. Associations between seropositivity and potential risk factors for EEHV infection in the final multivariable regression model the variable “region” was represented. Finally, publication of this thesis will increase professional and public awareness of the threats of TB and EEHV infection in Elephants in Thailand

Serological study on brucellosis in captive elephants (Elephas maximus) and stray dogs in North Thailand

Background and Aim: Brucellosis is considered as an important zoonotic disease caused by various strains of Brucella in numerous host species. Although brucellosis has been reported in almost animal species, the relevance of brucellosis infection and diagnostic technique in Asian elephant (Elephas maximus) has been limited. The present serological investigation aimed to investigate the antibody response to Brucella abortus in captive Asian elephants in North Thailand. Moreover, further serological survey was also conducted to detect the antibody response to Brucella canis in stray dogs cohabiting the same area as the elephant herd. Materials and Methods: Serum samples were collected from 40 captive Asian elephants and submitted for serological analysis based on B. abortus antigen using Rose Bengal plate test (RBPT) in combination with ethylenediaminetetraacetic acid-tube agglutination test (EDTA-TAT) as a supplementary test and by commercial indirect enzyme-linked immunosorbent assay (iELISA). In addition, serum samples were also obtained from 16 stray dogs that live nearby the elephant-raising area and were tested using commercial Dot-ELISA based on B. canis antigen. Results: Serological analysis in captive Asian elephants showed 100% seronegative (40/40) from all serological tests response to B. abortus. For stray dogs, 12.5% (2/16) had a low positive reaction response to B. canis. Conclusion: The serological survey for brucellosis in Asian elephant was adapted and applied using RBPT, EDTA-TAT, and iELISA in the present study. For future evaluation, we recommended the use of a combination of serological tests with validation together with comparing by direct detection such as bacterial isolation to provide an appropriate brucellosis surveillance program in Asian elephants. In addition, the surveillance of stray dogs or multispecies habitation should be kept into considerations

Serum Insulin-like Growth Factor-1 Is a Biomarker of Testosterone Production and Intact Acrosome in Asian Elephants (Elephas maximus)

The objective of this study was to find relationships among serum IGF-1, serum testosterone, seminal plasma IGF-1 concentrations and semen parameters in Asian elephants (Elephas maximus). A total of 17 ejaculates (one to three ejaculates/bull) were collected from seven captive elephant bulls by performing rectal massage. Before each ejaculation, blood samples were obtained for serum IGF-1 and testosterone assays. Subsequently, the semen characteristics of each ejaculate were evaluated. Mean serum IGF-1 concentration of elephant bulls was estimated as 326.3 &plusmn; 114.6 ng/mL (median, 286.2 ng/mL; range, 167.4&ndash;542.7 ng/mL). An increase in serum IGF-1 concentration was found to correlate with the percentage of spermatozoa with intact acrosomes. In addition, IGF-1 concentration was positively correlated with testosterone level. However, seminal IGF-1 concentrations could not be detected. In conclusion, our findings suggest that serum IGF-1 concentration is likely a biomarker of normal testicular functions, particularly spermatogenesis in elephants. Moreover, this commercial IGF-1 ELISA is eligible for analyzing serum IGF-1 concentration in Asian elephants

Seasonal Effects on Body Condition and Characteristics of the Estrous Cycle in Captive Asian Elephants (<i>Elephas maximus</i>) in Thailand: A Retrospective Study

The aim of this study was to investigate the effects of season on the body condition score (BCS), the characteristics of the estrous cycle (luteal phase [LPL], follicular phase [FPL], estrous cycle [ECL] lengths, and the start of the luteal phase [SLP] and follicular phase [SFP]), and progesterone levels (baseline and peak) of eight captive Asian elephants (Elephas maximus) in Thailand. From 2014 to 2019, blood samples were collected weekly for serum progesterone enzyme immunoassays (EIAs). Estrous cycles (n = 70), including the luteal and follicular phases, and BCS (n = 70) were recorded. Based on the BCS, the LPL, FPL, and ECL were assigned to the following two groups: normal (BCS = 3.0–4.0, n = 38) and overweight (BCS = 4.5–5.0, n = 32). The findings demonstrated that there was no difference in LPL between the groups. However, in the normal group, the ECL was one week longer (14.9 ± 1.7 vs. 13.9 ± 1.7 weeks; p p = 0.06) than in the overweight group. The mean progesterone level during the rainy, hot, and cool seasons was not statistically different. Based on the yearly averaged BCS from three seasons, the baseline and peak levels of progesterone were classified into the normal (n = 16) and overweight (n = 12) groups. Females with a normal BCS tended to exhibit higher progesterone peak levels (p = 0.08). The majority of peaks appeared during the rainy season (53.57%). The BCS was highest during the hot (4.47) and rainy (4.38) seasons, but not during the cool (4.12) season. The LPL, FPL, and ECL were not affected by the season in which the luteal phase occurred. On the other hand, the rainy season had a significant effect on the SFP, resulting in a longer LPL (p p = 0.01); both were the longest during the rainy season. In conclusion, the effects of season on BCS may be related to characteristics of the estrous cycle and peak progesterone levels. Ultimately, these findings provide ground knowledge to assist elephant managers and owners in planning breeding activities using seasonal effects and BCS measurements in tropical climates

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

Survival analysis of confirmed elephant endotheliotropic herpes virus cases in Thailand from 2006 - 2018.

The elephant endotheliotropic herpesvirus (EEHV) has been a known cause of death of young elephants in Thailand for over a decade. In this study, we report on the demography, disease characteristics and mortality of 58 elephants with confirmed EEHV hemorrhagic disease between January 2006 and August 2018 using retrospective data subjected to survival analysis. Median age of EEHV presentation was 29 months, and the mortality rate was 68.97% with a median survival time of 36 h. Most EEHV cases occurred in the north of Thailand, the region where most of the country's captive elephants reside. The hazard ratio analysis identified application of medical procedures and antiviral medications as being significant factors correlated to the risk of death. Our results indicate a need to focus EEHV monitoring efforts on young elephants and to follow current protocols that advise starting treatments before clinical signs appear