LH secretion and response to GnRH during seasonal anoestrus of the Pére David's deer hind (Elaphurus davidianus)

The pattern of LH secretion and response to exogenous GnRH was determined on 5 occasions during seasonal anoestrus of the Père David's deer hind. LH pulse frequency was low (3.3 +/- 0.6 pulses/18 h) in early anoestrus (February), increased significantly in mid-anoestrus (April; 8.4 +/- 1.4 pulses/18 h) and thereafter declined slightly in late anoestrus (June; 6.3 +/- 0.25 pulses/18 h). Mean LH concentrations also showed significant changes during anoestrus with higher levels in mid-anoestrus (April; 0.85 +/- 0.12 ng/ml) when compared with other times (0.53 +/- 0.05, 0.60 +/- 0.10, 0.68 +/- 0.06 and 0.71 +/- 0.05 ng/ml for February, March, May and June, respectively). LH pulse amplitude showed no significant changes during the study. The LH response to intravenous injections of 2 micrograms GnRH was lowest in early anoestrus (February), increased significantly in mid-anoestrus (April) and remained high through late anoestrus. The response during the luteal phase was similar to that seen during late anoestrus. These results indicate that seasonal anoestrus in the Père David's deer hind is not a uniform state but is characterized by an early period of 'deep' anoestrus.     

A QTL study of growth and body shape in the inter-species hybrid of Père David's deer (Elaphurus davidianus) and red deer (Cervus elaphus)

An interspecies deer hybrid resource population developed from a cross of Père David's and red deer was used to detect QTL that account for species differences. A genome scan, coupled with composite interval mapping, was conducted to search for QTL controlling body measurements at pre-pubescent age (6 months of age) and puberty (15 months of age) in this interspecies hybrid. Five linkage groups that harbour QTL affecting morphology were identified. A joint-traits analysis was used to search for putative pleiotropic QTL on four of these linkage groups, and three were significantly associated with pleiotropic QTL for nose width and foot length (metacarpal and phalanges), which collectively accounted for 29-58% of the phenotypic difference between the two deer species. This study suggests that a few loci with large pleiotropic effects may be responsible for species-specific differences in growth and structure-related traits.     

Seasonal home range patterns of the reintroduced and rewild female Père David’s deer Elaphurus davidianus

To learn the seasonal home range pattern and its role in the ecological adaptation of rewild Père David’s deer, we took the investigation from spring to winter in three-collared females that released into the coastal wetlands of the Yellow Sea, China. Using GPS collars, minimum convex polygons and fixed kernel estimation, we recorded the movement and estimated the home ranges of the deer. The results showed that the collared deer inhabited only 42.85% of the coastal wetland habitat that had been designated for them. The home ranges were larger during winter and spring than in autumn and summer. In summer and autumn, the collared deer remained in the reserve, which was dominated by spartina grass. In the spring and winter, they were lured by the green leaves of wheat in the farmlands outside the reserve but were then driven back by people. Home range patterns reflected the seasonality of the availability of food resources and human activity. In addition to the frequently utilized habitats, much larger wetland areas are required for such deer. As the population density increases gradually, more available space will be needed to meet the fundamental needs of the seasonal dispersal of rewild Père David’s deer.     

cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.     

Seasonal changes of reproductive behavior and fecal steroid concentrations in Père David's deer.

From July 1997 to September 1997 and from March 1998 to July 1998, we studied reproductive behaviors of Père David's deer in Dafeng, China. During the field behavioral observations, we collected fresh voided fecal samples from the Père David's stags and hinds periodically and kept those samples under -20 degrees C until laboratory analysis. We analyzed the fecal testosterone, estradiol, and progesterone concentrations in those samples using radioimmunoassay. During this study, we also recorded 17 types of male reproductive behaviors and nine types of female reproductive behaviors. Reproductive behaviors and the fecal steroid concentrations showed overt seasonal fluctuations. There were statistically significant correlations between some male reproductive behaviors, such as anogenital sniffing, urine sniffing, urine spraying, wallowing, bellowing, antler adorning, Antler swags mud, chasing, herding hinds, chin resting, mounting and copulating, with the fecal testosterone concentrations. These results suggested that seasonal reproductive behaviors in stags are strongly associated with circulating testosterone. We also found that some female reproductive behaviors fluctuate corresponding with changes in fecal estrogen concentrations. Although there was no direct evidence to confirm the correlations between female reproductive behaviors and fecal estrogen in our experiment, we could not rule out that reproductive activities of hinds were largely related to ovarian estrogen secretion, and estrogen is necessary for inducing female reproductive behaviors.     

Diet composition and selection of Père David's deer in Hubei Shishou Milu National Nature Reserve,China

Hubei Shishou Milu National Nature Reserve is an ideal place to restore the wild population of Père David's deer (Elaphurus davidianus). Understanding foraging ecology and diet composition is essential for assessing population development or establishing long-term effective conservation measures for endangered species. However, little is known about the diet composition of Père David's deer and its diet selection mechanism. In this study, we used stable isotope technology to investigate the diet composition of Père David's deer according to various tissues (i.e., fur, muscle, liver, heart, and feces) and seasons, and evaluated the correlation between the nutrient composition of plants and diet composition. Bayesian isotope analysis showed that the autumn and winter diet estimated by fur and fecal samples indicated a diet dominated by C₃ grasses (42.7%–57.2%, mean), while the summer diet estimated by muscle and liver samples was dominated by C₃ forbs (30.9%–41.6%, mean). The Pearson correlation test indicated that the contribution of winter diet composition reflected by fur and fecal samples was associated with correlations with crude protein (r = .666, p < .01) and soluble sugars (r = .695, p < .01). The results indicated that crude protein and soluble sugars were important factors influencing the winter diet selection of Père David's deer. In the context of the current reintroduction facing many challenges, such as habitat fragmentation, wetland degradation, and human disturbance, comprehensively evaluating the diet selection mechanism of Père David's deer under different resource specificities and temporal changes should be considered in the future.     

QTL for pubertal and seasonality traits in male Père David's x red deer interspecies hybrids

The unique Pere David's (Elaphurus davidianus) x red deer (Cervus elaphus) backcross hybrid has been used to search for evidence of quantitative trait loci (QTL) for antler pubertal (date and live weight at pedicle initiation) and antler seasonality (date of antler cleaning and casting) traits in temperate species of deer. Analyses using marker information revealed evidence for a QTL for date at pedicle initiation (LOD = 3.7) and live weight at pedicle initiation (LOD = 3.1). These QTL explained 13% and 11% of the phenotypic variance in these traits, respectively.     

QTL for live weight traits in Père David's x red deer interspecies hybrids

Interspecies hybrids between Père David's deer (Elaphurus davidianus) and red deer (Cervus elaphus) have proved to be a powerful resource in the search for quantitative trait loci (QTL) in deer. Several regions of the genome with significant effects on live weight and growth rates in backcross hybrids were detected. These include putative QTL for 6-month live weight (LOD 3.90) on linkage group 12, for 14-month live weight (LOD 3.19) on linkage group 1, three putative QTL for growth rate from 3 to 6 months (LOD 4.19 on linkage group 12, LOD 3.92 on linkage group 12, and LOD 3.34 on linkage group 5). In addition, linkage groups 20 and 1 appear to be associated with live weight traits between 9 and 16 months. The variance in traits explained by these QTL ranged between 5.3% and 11.2%. Allele substitution with Père David's alleles at different loci had both positive and negative effects on live weights and growth rates.     

A novel HURRAH protocol reveals high numbers of monomorphic MHC class II loci and two asymmetric multi-locus haplotypes in the Père David's deer

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ～ DRB1 ～ DRB3 ～ DQA1 ～ DQB2 (H1) and DRA1*02 ～ DRB2 ～ DRB4 ～ DQA2 ～ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.     

Do Père David's Deer Lose Memories of Their Ancestral Predators?

Whether prey retains antipredator behavior after a long period of predator relaxation is an important question in predator-prey evolution. Père David''s deer have been raised in enclosures for more than 1200 years and this isolation provides an opportunity to study whether Père David''s deer still respond to the cues of their ancestral predators or to novel predators. We played back the sounds of crows (familiar sound) and domestic dogs (familiar non-predators), of tigers and wolves (ancestral predators), and of lions (potential naïve predator) to Père David''s deer in paddocks, and blank sounds to the control group, and videoed the behavior of the deer during the experiment. We also showed life-size photo models of dog, leopard, bear, tiger, wolf, and lion to the deer and video taped their responses after seeing these models. Père David''s deer stared at and approached the hidden loudspeaker when they heard the roars of tiger or lion. The deer listened to tiger roars longer, approached to tiger roars more and spent more time staring at the tiger model. The stags were also found to forage less in the trials of tiger roars than that of other sound playbacks. Additionally, it took longer for the deer to restore their normal behavior after they heard tiger roars, which was longer than that after the trial of other sound playbacks. Moreover, the deer were only found to walk away after hearing the sounds of tiger and wolf. Therefore, the tiger was probably the main predator for Père David''s deer in ancient time. Our study implies that Père David''s deer still retain the memories of the acoustic and visual cues of their ancestral predators in spite of the long term isolation from natural habitat.     

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.     

Eighteen novel polymorphic microsatellite loci developed from the Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>)

Père David’s deer is a severely bottlenecked species but without showing inbreeding depression, making it essential to develop molecular markers to explore her genetic mechanism of population recovery. In this study, we isolated 18 novel polymorphic microsatellite loci from a dinucleotide-enriched library. This suit of markers presented 2–3 alleles for each locus and their observed and expected heterozygosities were 0.057–0.610 and 0.056–0.598, respectively. These new microsatellite loci had an average of 2.12 alleles and thus contributed to relatively low exclusion probabilities of parentage and paternity testing (0.768 and 0.921). However, when these loci were examined in combination with previous microsatellite markers, overall probabilities of parentage and paternity exclusion went up to 0.905 and 0.990, respectively, showing that these 26 microsatellite loci should be adopted together in future genetic analyses for this highly inbred species.     

Advertisement calls of Guenther’s frog Hylarana guentheri (Anura: Ranidae) during the breeding season

Acoustic signalling is the most important form of communication in anuran amphibians. Here we recorded and analysed the calls of 18 male Guenther’s frogs (Hylarana guentheri) from the wild during the breeding season. The advertisement calls of H. guentheri were composed of from a single note to five notes, with three-note calls the most recorded. All individuals produced calls around 600 Hz but calls ranged from 470 to 2600 Hz. Comparing the differences between individuals calls, we found within-male coefficients of variation (CV_w) of call intensity, the fundamental frequency, the first formant, the second formant, the third formant and the fourth formant were static (less than 5% variation), whereas those of note duration, call duration, call interval, numbers of pulses and dominant frequency were dynamic (larger than 15% variation). Comparisons of the call characteristics of H. guentheri in this study with other studies from China, Singapore and Vietnam found call characteristics varied greatly between the five different locations.     

Genetic variability in relocated Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>) populations—Implications to reintroduction program

Since 1985, China has established three breeding herds of Père David’s deer: the Beijing Père David’s Deer Park (39°07′N, 116°03′E), the Dafeng Père David’s Deer Nature Reserve (33°05′N, 120°49′E) and Shishou (Tianezhou) Père David’s Deer Nature Reserve (29°49′N, 112°33′E), through reintroductions of about 30–40 founders. Since establishment, all three populations have grown steadily. However, genetic backgrounds in those populations are still unknown. We studied the genetic diversity in Père David’s deer and genetic consequences of population relocations in China. We revealed that genetic diversity was extremely low in Père David’s deer populations in China. Only a single mtDNA D-loop haplotype was found in the deer, furthermore, only five polymorphic microsatellite loci were screened out from 84 pairs of species-transferred primers. Genetic makeup in the three Père David’s deer populations were significantly different (P < 0.01). H _E and allelic richness in the Tianezhou population were the highest (0.54, 2.60, n = 31), Beijing population (0.52, 2.4, n = 125) showed the second highest measures, while the Dafeng population (0.46, 2.39, n = 39) measured lowest. Our results suggest that effective management of a species of low genetic diversity like the Père David’s deer should consider the genetic background of each founder to make sure genetic variations are preserved in both source population and relocated population. Now, the Tianezhou population is the most appropriate source population in China when establishing new Père David deer populations in the wild.

The “males earlier than females” phenomenon in the fawning season of fallow deer (Dama dama)

Twenty years ago, Gaede (in Hemmer, Nutztier Damhirsch Rheinischer Landwirtschafts-Verlag, Bonn, pp 95–112, 1986) pointed to a trend that male fawns of farmed fallow deer were born predominantly within the first half of the fawning season, whereas female fawns were born primarily during the second half. The data basis collected during 25 years of pedigree breeding in the fallow deer domestication project directed by the author shows this “males earlier than females” phenomenon to be real and statistically highly significant. It seems to depend on the age of the siring bucks and may mirror the “fat-season-syndrome” during the rut.This paper is dedicated to Dr. agr. Ernst-Adolf Gaede, former director of the Lehr- und Versuchsanstalt für Viehhaltung Neumühle (Münchweiler an der Alsenz), for his 80th birthday on July 15, 2006.     

Assaying progesterone,estradiol and cortisol concentrations in hair of Père David deer hinds: an alternative way to reflect seasonality of steroid secretion

The use of a hair hormone concentration assay is increasingly recognized as a useful and noninvasive technique for monitoring the endocrinological status of animals. However, few studies have focused on reproductive and stress hormones together. We used a chemiluminescent immunoassay to determine whether the progesterone, estradiol, and cortisol concentrations could be measured from hair and whether these hormone concentrations varied in different hair segments of captive Père David deer hinds. We found that progesterone, estradiol, and cortisol could be measured in the hair samples and that the progesterone concentration varied but the estradiol and cortisol concentrations did not among different hair segments. Contrary to the segmental decline in hair cortisol found in many studies, we found that progesterone concentration was higher near the tip than at the base of hair in Père David deer. This suggests that the variation in segmental hair steroid hormone concentration in seasonal molting animals may be mainly due to internal reproductive cycles and that hair steroid hormones may reflect long-term physiological changes and can thus be used for the conservation and management of wildlife.