Semen characteristics and second successful artificial insemination of Asian elephant (Elephas maximus) in Thailand.

Background and Aim: As the number of wild Asian elephants (Elephas maximus) continues to decline, maintaining healthy populations under human care is vital. Male fertility assessment is essential for understanding the reproductive status, which can help to uncover underlying problems and improve the rate of pregnancy success. The objectives of this study in Asian elephants were as follows: (1) To investigate the semen characteristics; (2) to compare the relative seminal vesicle size and semen characteristics; (3) to compare the semen characteristics between good-motile (>60% progressive motility) and poor-motile (<60% progressive motility) ejaculates; and (4) to investigate the pregnancy success rate after artificial insemination (AI) with combined chilled and frozen semen. Materials and Methods: In total, 153 ejaculates were collected by manual rectal stimulation from 25 bulls. The volume, pH, sperm concentration, progressive motility, viability, morphology, and membrane integrity were investigated in each ejaculate. Assessment of accessory sex glands was conducted using transrectal ultrasonography to compare the relative seminal vesicle size and semen characteristics, and the bulls were divided into two groups according to the size of the ampulla (<7 or ≥7 cm2). For the comparison of good and poor-motile ejaculates and semen characteristics, the samples were divided into two groups: Good-motile (>60% progressive motility) and poor-motile (<60% progressive motility) ejaculates. Semen ejaculates for AI were collected from three bulls. The estrous cycles of four females were monitored using an enzyme immunoassay. Seven AI attempts were conducted using frozen and/or chilled semen by endoscopic visualization. AI was repeated 1 day before the luteinizing hormone surge, on the day of the surge, and 1 day after the surge. Pregnancy was confirmed by monitoring the serum progesterone profile and the abdomen and mammary glands changes. Results: From 153 ejaculates, the mean±standard error values of progressive motility, semen volume, sperm concentration, pH, and viability were 40.18%±2.28%, 40.94±3.86 mL, 1,205.58±62.26×106 sperm/mL, 7.50±0.10, and 56.17%±1.96%, respectively. Comparing ampulla size and semen characteristics revealed that the bulls with ampullae of ≥7 cm2 yielded significantly larger volume ejaculates. However, there were no significant differences in sperm motility and concentration. The comparison of semen characteristics between good- and poor-motile ejaculates revealed that the former had significantly higher pH, viability, normal acrosomes, intact membranes, and normal head and tail morphology but often had a significantly lower volume and sperm concentration. From seven AI attempts in four females, one female had a confirmed pregnancy (14.3% pregnancy rate), and delivered a healthy live female baby weighing 128 kg at 21 months and 12 days of gestation. The baby is now 3 years old and in a healthy condition, with normally developing growth and behavior. Conclusion: The semen characteristics of Asian elephants can be used as the baseline reference for further applications. The ampullae size indicates semen quantity but not quality. Our success in producing an elephant calf from AI using frozen and chilled semen demonstrated that AI can be used as an alternative approach for the breeding management of Asian elephants. However, the semen of Asian elephants is of poor quality, especially in terms of membrane integrity; thus, the improvement in semen quality through intensive and careful management of elephant health and fertility remains a challenge for the future. Furthermore, a sperm bank should be established to develop sperm cryopreservation, which will be invaluable for improving the genetic diversity of the Asian elephant.

The complete mitochondrial genome of Fea's muntjac (Muntiacus feae Thomas and Doria, 1889) with phylogenetic analysis†.

The complete mitochondrial genome (mitogenome) of the rare Fea's muntjac (Muntiacus feae) was sequenced (GenBank accession nos. MG857662-MG857664). The mitogenome was found to be 16,355 bp in length with base compositions of 33.16% A, 24.59% C, 13.46% G, and 28.78% T and a GC content of 38.06%. The genome is comprised of 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop). Phylogenetic analysis revealed that Fea's muntjac is more closely related to Black muntjac (M. cronifrons) than to Red muntjac (M. muntjak). These data will be useful for further studies on the genetic diversity and molecular phylogenetic relationship of the genus Muntiacus.

Hypervariability of Nucleolus Organizer Regions in Bengal Slow Lorises, Nycticebus bengalensis (Primates, Lorisidae).

Slow lorises are a cryptic species complex, and thus genetic markers are needed to identify distinct evolutionary lineages or species. We examined the nucleolus organizer regions (NORs) of Bengal slow lorises (Nycticebus bengalensis) using FISH with 18S rDNA (rDNA-FISH) and silver nitrate staining (Ag-NOR stain). Ten individuals of the putatively single species N. bengalensis showed higher variability in localization than 3 other congeners, though their overall karyotypes were similar. The rDNA-FISH analysis detected a total of 18 loci, in contrast to previous studies of other slow loris species that revealed far fewer (6-10) loci. Eight of the 18 loci detected in the present analysis were found to be semi-stable localizations at 4 different chromosomes, while 10 were found to be unstable localizations at 5 other chromosomes. The semi-stable locations showed occasional presence/absence of variations for rDNA-FISH, and unstable locations were polymorphic among individuals, contributing to the higher variability of NORs in this taxon. We hypothesize that the larger numbers of rDNA loci found in N. bengalensis were introduced by genomic dispersion through ectopic recombination in association with terminal regions including rDNA. Such differences are potentially very powerful chromosomal markers to be used in species identification and conservation.

Body mass dynamics in hand reared clouded leopard (Neofelis nebulosa) cubs from birth to weaning.

To study the dynamics of body mass changes in hand reared clouded leopards, we analyzed 3,697 weight data points during the first 3 months of life in 49 cubs from 24 zoo-born litters from 2003 through 2012. All cubs were fed the same formula mixture after a similar weaning protocol. The hand rearing process was divided into three periods based on feeding protocols: Stage 1: formula only (Days 1-28; Day 0 = day of birth); Stage 2, formula supplemented with protein (e.g., turkey baby food; Days 29-42); Stage 3, formula in decreasing amounts supplemented with meat (chicken and/or beef; Days 43-90). Weights at birth were 11.2% higher (P < 0.001) for males (n = 29) than females (n = 20). Daily weight gain was slowest (P < 0.05) during Stage 1 when cubs were fed straight formula only and fastest during Stage 3 when provided a mixture of formula and meat. Mean growth rate (± SD) during hand rearing differed (P < 0.05) by gender, being 34.6 ± 1.4 g/day for male and 30.0 ± 1.2 g/day for female cubs. Eighteen cubs (37%) exhibited mild to severe diarrhea during the study; however, palliative treatment resulted in similar (P > 0.05) growth and weaning weights compared to healthy counterparts. These are the first data documenting, on a large scale, the growth patterns for zoo born, hand reared clouded leopard cubs. Findings are valuable as an aid in managing this rare species, including for helping identify early onset of medical issues and further determining key factors regulating the first 3 months of development.

Swimmer syndrome in a clouded leopard (Neofelis nebulosa) cub.

A 32-day-old, hand-reared, captive-born female clouded leopard (Neofelis nebulosa) cub presented as being unable to stand, ambulate, or adduct both hind limbs. The cub exhibited hyperextension of both tarsal joints and a flattened thorax, which limited mobility to "swimmer-like" movements. Neither congenital defects nor neurologic deficits were observed during the medical examination. Radiographic examination showed the thorax was compressed dorsoventrally, but no other skeletal abnormalities were detected. Based on clinical signs, the condition was more consistent with swimmer syndrome, which has been described in young offspring of several domestic species. Over the course of 3 wk, affected limbs were treated by intensive physiotherapy, corrective bandages were applied, and thermotherapy was used to improve circulation, which resulted in a complete recovery and development of subsequent normal ambulation. It is concluded that early diagnosis and treatment of this condition led to the resolution of clinical signs, resulting in normal development of the clouded leopard cub reported here.

Captive breeding and reintroduction of the oriental pied hornbill (Anthracoceros albirostris) in Khao Kheow Open Zoo, Thailand.

This article discusses two related issues of the captive breeding and reintroduction of the Oriental pied hornbill (Anthracoceros albirostris) in order to increase its population in the natural habitats. Oriental pied hornbills were bred in Khao Kheow Open Zoo. Three pairs were separated and kept in breeding cages. Females occupied artificial nests between February and April 2005-2007. Eggs were laid and incubated between February and March each year from 2005 to 2007. Nestlings hatched in late March and left the nest in late April 2005-2007. Each breeding pair was fed with approximately 400 g of food each day. All three pairs reproduced resulting in mature offspring of seven in 2005, six in 2006, and five in 2007. Four of sixteen 3-year-old birds were randomly selected and equipped with a GPS receiver on their backs. Activities of the birds attached and unattached with GPS were not significantly different. The first two birds (one female, one male with GPS) were reintroduced on August 5, 2006, and another nine birds (four birds with GPS) were reintroduced on December 26, 2006. The average home range of these reintroduced birds was 0.13 km(2) . Their foods consisted of wild plants and animals in the home range. The first reintroduced pair was able to breed naturally by laying and hatching eggs in an artificial nest. Two juveniles left the nest in April 2008. These results indicate that both captive breeding and reintroduction are potentially important ways to increase the population of the Oriental pied hornbill in natural habitats.