Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon)

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Malignant melanoma in a captive red deer (Cervus elaphus elaphus)

We report a case of malignant melanoma in a captive red deer (Cervus elaphus elaphus). The primary lesion was on the plantar aspect of the right pastern and infiltrated the surrounding skin, but not underlying tendons or bone. Diffuse metastasis to the regional lymph nodes had occurred. Histologically, lesions were characterized by large numbers of variably pigmented epithelioid cells with a high degree of nuclear atypia and many mitotic figures. This is the first reported case of this neoplasm in red deer.     

Phylogeography of red deer (Cervus elaphus) in Europe

Aim To investigate the phylogeographical patterns of red deer (Cervus elaphus) in Europe, and to disentangle the influence of ancient (e.g. Pleistocene ice ages) from more recent processes (e.g. human translocations). Location Europe. Methods In this study we provide by far the most extensive analysis of genetic structure in European red deer, based on analyses of variation at two mitochondrial markers (cyt b and D‐loop) in a large number of individuals from 39 locations. Relationships of mitochondrial DNA haplotypes were determined using minimum spanning networks and phylogenetic analyses. Population structure was examined by analyses of molecular variance. Historical processes shaping the present patterns were inferred from nested clade analysis and nucleotide diversity statistics. Results Within Europe, we detected three deeply divergent mitochondrial DNA lineages. The three lineages displayed a phylogeographical pattern dividing individuals into western European, eastern European and Mediterranean (Sardinia, Spain and Africa) groups, suggesting contraction into three separate refugia during the last glaciation. Few haplotypes were shared among these three groups, a finding also confirmed by F_ST values. Calculations of divergence times suggest that the groups probably split during the Pleistocene. Main conclusions The observed pattern is interpreted to result from isolation in different refugia during the last glaciation. The western and eastern European lineages could be linked to an Iberian and Balkan refugium, respectively. The third lineage might originate from a Sardinian or African refugium. We link local phylogeographical patterns observed in Europe to the post‐glacial recolonization process, shaped by the geographical localization of refugia and barriers to gene flow. Regardless of the importance of red deer as a game species and the tradition of translocating red deer in Europe, we detected few individuals that did not match the trichotomous pattern, suggesting that translocations have occurred mainly at smaller spatial scales.     

Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus)

Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.     

Mitochondrial DNA phylogeography of red deer (Cervus elaphus)

In order to understand the origin, phylogeny, and phylogeography of the species Cervus elaphus, we examined the DNA sequence variation of the mitochondrial cytochrome b gene of 51 populations of deer from the entire distribution area of Cervinae with an emphasis on Europe and Asia. Several methods, including maximum parsimony, maximum likelihood, and nested clade analysis, revealed that red deer originated from the area between Kyrgyzstan and Northern India. We found two distinct groups of red deer: a western group consisting of four subgroups and an eastern group consisting of three subgroups. Our mtDNA data do not support the traditional classification of red deer as only one species nor its division into numerous subspecies. The discrepancies between the geographical pattern of differentiation based on mtDNA cytochrome b and the existing specific and subspecific taxonomy based on morphology are discussed.     

Onchocercosis in red deer (Cervus elaphus) from Spain.

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of P. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection +/- SD was 3.93 +/- 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer.     

Freemartinism in red deer (Cervus elaphus L.)

Several mixed-sex twins were produced by red deer treated with progesterone and pregnant mare's serum gonadotrophin. Of seven females karyotyped, two were 68,XX/68,XY chimeras. One male had the same chimeric karyotype as its female co-twin. These are the first reported cases of freemartinism in deer.     

Genetic structure of the Danish red deer (Cervus elaphus)

The red deer (Cervus elaphus) population in Denmark became almost extinct in recent historical times due to over‐hunting. The species has subsequently recovered within remote areas, but non‐Danish individuals have been introduced at several localities. To assess genetic structure, past demographic history, and the possibility of a still existing original stock, we analysed 349 specimens from 11 geographically separate areas and from three enclosed areas, genotyping 11 microsatellite loci. Moreover, an 826‐bp fragment of the control region of the mitochondrial DNA was sequenced for 116 recent specimens and seven museum specimens. There was a significant difference in mean expected heterozygosity (H_E) between the three enclosed areas and the 11 unenclosed areas. Significant departures from Hardy–Weinberg equilibrium were observed in the three enclosed areas and in nine of the unenclosed areas. The overall degree of genetic differentiation among all 14 areas was significant (F_ST = 0.09, P < 0.01), primarily because the mean pairwise F_ST for the three enclosed areas was significantly higher than that for the 11 unenclosed areas. A Bayesian clustering procedure detected three genetically distinct populations and indicated reduced gene flow between the enclosed and unenclosed areas. The individuals in the unenclosed areas show genotypic mixture, presumably as a result of gene flow among them. Markov Chain Monte Carlo simulations, based on the genealogical history of the microsatellite alleles, suggest a drastic decline in the effective population size of the enclosed areas some 188–474 years ago. Mitochondrial DNA analysis of the recent specimens showed seven haplotypes. Individuals from the enclosed Jægersborg Dyrehave contain haplotypes that occur all over Denmark and also are found in Western Europe. A close relationship between Scandinavian and Western European red deer is most likely. Only individuals from the unenclosed Lindenborg Estate and the enclosed Tofte Skov did not group with any other Danish individuals. As six of seven museum specimens had haplotypes also found in modern Danish samples, the current population of red deer in Denmark is genetically close to the original Danish red deer. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 688–701.     

Morphological observation of antler regeneration in red deer (Cervus elaphus)

Deer antler offers a unique opportunity to explore how nature solves the problem of mammalian appendage regeneration. Annual antler renewal is an example of epimorphic regeneration, which is known to take place through initial blastema formation. Detailed examination of the early process of antler regeneration, however, has thus far been lacking. Therefore, we conducted morphological observations on antler regeneration from naturally cast and artificially created pedicle/antler stumps. On the naturally cast pedicle stumps, early antler regeneration underwent four distinguishable stages (with the Chinese equivalent names): casting of previous hard antlers (oil lamp bowl), early wound healing (tiger eye), late wound healing and early regeneration (millstone), and formation of main beam and brown tine (small saddle). Overall, no cone-shaped regenerate, a common feature to blastema-based regeneration, was observed. Taken together with the examination on the sagittal plane of each regenerating stage sample, we found that there are considerable overlaps between late-stage wound healing and the establishment of posterior and anterior growth centers. Observation of antler regeneration from the artificially created stumps showed that the regeneration potential of antler remnants was significantly reduced compared with that of pedicle tissue. Interestingly, the distal portion of a pedicle stump had greater regeneration potential than the proximal region, although this differential potential may not be constitutive, but rather caused by whether or not pedicle antlerogenic tissue becomes closely associated with the enveloping skin at the cut plane. Antler formation could take place from the distal peripheral tissues of an antler/pedicle stump, without the obvious participation of the entire central bony portion. Overall, our morphological results do not support the notion that antler regeneration takes place through the initial formation of a blastema; rather, it may be a stem cell-based process.     

Reproductive seasonality in female Iberian red deer (Cervus elaphus hispanicus)

This study characterized the seasonal pattern of luteal cyclicity in Iberian red deer (n=16), by measuring plasma progesterone concentrations in hinds (female red deer) twice per week from calving (May and June) 1996 until May 1997. In eight of these hinds we also examined plasma prolactin profiles to assess seasonal responses to photoperiod. Plasma progesterone concentration in the 16 hinds studied indicated that the reproductive pattern is seasonal, and lasts for 5.73 +/- 0.27 months. After calving, progesterone levels remained basal (no luteal activity) for several months, except in a hind that lost her calf just after calving, and thus did not have to suckle it. This hind showed two consecutive estrus cycles in the month following calving, which suggests that suckling has an inhibiting effect on the resumption of ovarian activity. These results also showed that as long as the hinds do not become pregnant, they show between 5 and 10 estrus cycles per reproductive season (8.06 +/- 0.35), ranging between 105 and 249 days from onset of the first cycle to end of the last one. Uninterrupted cycling lasted for 3.5-6.4 months (mean, 4.6 +/- 0.24). Cyclic luteal activity was found from October to February in all hinds, with a smaller, but notable proportion in September (56.25%) and March (68.8%), whereas it was negligible in the remaining period. Our results show a reproductive season similar to or longer than that recorded by other authors. Prolactin plasma concentrations showed a yearly trend following that of photoperiod, with peak concentrations from April to July, a decrease in August, minimal concentrations from September to February and a sharp increase in March.     

Physiological effects of hunting red deer (Cervus elaphus).

When red deer (Cervus elaphus) were hunted by humans with hounds the average distance travelled was at least 19 km. This study of 64 hunted red deer provides the first empirical evidence on their state at the time of death. Blood and muscle samples obtained from hunted deer after death were compared with samples from 50 non-hunted red deer that had been cleanly shot with rifles. The effects on deer of long hunts were (i) depletion of carbohydrate resources for powering muscles, (ii) disruption of muscle tissue, and (iii) elevated secretion of beta-endorphin. High concentrations of cortisol, typically associated with extreme physiological and psychological stress, were found. Damage to red blood cells occurred early in the hunts; possible mechanisms are discussed. Taken together, the evidence suggests that red deer are not well-adapted by their evolutionary or individual history to cope with the level of activity imposed on them when hunted with hounds.     

Induction of early breeding in red deer (Cervus elaphus) by melatonin

Lactating (N = 12) and non-lactating (N = 6) red deer hinds and one stag at pasture were given concentrates (500 g/head) containing melatonin (5 mg/head) daily at 16:00 h from 18 June to 16 October. The stag shed the antler velvet and started rutting 5 weeks ahead of untreated stags, and hinds had their first oestrus and ovulation of the breeding season in mid-September, 5 weeks in advance of control lactating (N = 9) and non-lactating (N = 5) hinds. Treated hinds were allowed to mate with the treated stag and control hinds ran in an adjacent paddock with an untreated stag. All hinds became pregnant during the study with all but 2 melatonin-fed hinds (1 lactating, 1 non-lactating) and 1 control hind (lactating) conceiving at the first oestrus. Melatonin-treated hinds lost slightly more live weight than did controls from June to November, but their suckled calves grew throughout at a rate similar to those of control hinds.     

Long-term density-dependent changes in habitat selection in red deer (Cervus elaphus)

Understanding how habitat selection changes with population density is a key concept in population regulation, community composition and managing impacts on biodiversity and ecosystem services. At low density, it is expected that individuals select habitats in terms of their preference, but as population density increases, the availability of resources per individual declines on preferred habitats, leading to competition which forces some individuals to exploit less preferred habitats. Using spatial information of Scottish red deer (Cervus elaphus) winter counts, carried out in 110 areas across Scotland between 1961 and 2004 (a total of 1,206,495 deer observations), we showed how winter habitat niche breadth in red deer has widened with increasing population density. Heather moorland and montane habitats were most and least preferred for deer, respectively. Increasing density favoured the selection of grassland, to the detriment of the selection of heather moorland. The selection of heather and grassland decreased when temperature increased, while the selection of montane and peatland habitats increased. These findings are important for understanding how habitat use, density and population are likely to be affected by weather, and allow us to predict habitat impacts by large mammal herbivory and climate.     

Melatonin can induce year-round ovarian cyclicity in red deer (Cervus elaphus)

From 17 February 1987 (Day 1) to 5 June 1988 (Day 475), 6 red deer hinds which had been in natural daylength (NL/M) and 6 hinds which had been in continuous artificial light for the previous month (CL/M) were each given melatonin (5 mg in feed) daily at 15:00 h. Six controls (C) received unsupplemented feed. From Day 1 all hinds were in natural daylight and ovarian cyclicity was assessed from plasma progesterone concentrations. Group C first went into anoestrus on 15 March 1987 (Day 27 +/- 9.2 (s.e.m], recommenced cyclicity on 23 October (Day 249 +/- 2.3) and went into anoestrus again on 2 April 1988 (Day 411 +/- 8.7). Group CL/M first went into anoestrus 31 days earlier (P less than 0.05) on 12 February (Day -4 +/- 7.8), before the start of melatonin treatment; 4 hinds then recommenced ovarian cycles 132 days earlier (P less than 0.001) on 13 June (Day 117 +/- 5.8) and continued to cycle for a longer period than did controls. Group NL/M hinds were cyclic at the start of melatonin feeding and continued to cycle for 1 year or more (N = 6). Plasma prolactin concentrations remained suppressed (less than 20 ng/ml) for the duration of melatonin-feeding (Groups CL/M and NL/M) whereas control values (Group C) were elevated (20-120 ng/ml) between April and August (P less than 0.05). The ovarian response by hinds to melatonin therefore depends on initial reproductive status and recent photoperiodic history, and continued administration to cyclic hinds stimulates prolonged ovarian cyclicity irrespective of the time of year.     

Quantifying the damage of red deer (Cervus elaphus) grazing on grassland production in southeastern Slovenia

The influence of red deer (Cervus elaphus) grazing on grassland production for forage conservation at the forest border during the vegetation period was studied on three locations (Mala gora, Cvišlerji, and Mačkovec) in the Kočevje region (SE Slovenia). The experiment lasted from the 25th of March until the 8th of October 2002. Portable cages were used to exclude red deer from grazing the herbage. At four sampling dates in the season, herbage air dry matter (DM) yield was measured at three different observations (cage-protected plot, cage-protected plot only two to three weeks before sampling date, otherwise freely grazed (removed), and unprotected plot). The results from the experiment showed that red deer grazed on grassland through the entire season and that the regeneration capability of sward was the highest in summer, middle in spring and smallest in autumn. On unprotected plots, an average reduction of 50% of herbage DM yield was found with the most damaged sites also up to 80% reduction.