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.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

Effect of reproductive rest on the subsequent breeding in Iberian red deer hinds (Cervus elaphus hispanicus)

The objective of this study was to examine the effects of reproductive rest during the preceding year on subsequent breeding in captive Iberian hinds. To achieve this, influences were assessed on hind weight at mating, precalving and postcalving weight, hind body condition at mating, hind weight gain during gestation, hind weight loss at calving, total milk yield and calf birth weight during the following breeding season. Sex ratio was examined according to hind age class (sub-adults or adults), precise age, body condition and weight at mating, and reproductive rest. One hundred and twenty seven gestations and their subsequent lactations were analyzed in pluriparous Iberian red deer (Cervus elaphus hispanicus) kept in captivity for two years. Sixty four of these gestations (50.4%) took place after no gestation during the preceding year.Hinds that did not gestate the preceding year were heavier at mating and after calving, gained less weight during gestation, and their body condition at mating was better than those hinds that had gestated. Also, calves from hinds that had reproductive rest the preceding year were heavier at birth than the others. Nevertheless, the probability of bearing a male was greater with increasing age, but not with reproductive rest or any other variable. Total milk yield was positively affected by hind postcalving weight and hind age at mating, but not by reproductive rest.In conclusion, reproductive effort during the preceding breeding influenced the subsequent gestation, having an effect on the following variables: hind weight and body condition at mating, hind weight gain during gestation, postcalving weight, and calf birth weight.     

Lactation under induced five-month delay reproduction in Iberian red deer (Cervus elaphus hispanicus)

In Iberian deer, calf birth date affects both milk production and composition, as well as the weight change of hind during lactation and calf growth. This study examines a five-month induced delay in calving (autumn group) in a group of 19 Iberian hinds compared to a group of 13 standard calving hinds (spring group). Fertility in the autumn group was half that of the spring one. From the former, a further two calves out of six died before or soon after birth. Successfully lactating hinds showed a greater concentration in protein, fat and lactose (P < 0.001), but did not differ from spring group in total milk yield, calf gain or weight change of hinds during lactation. However, after correcting for the greater calf or hind body weight at calving of the autumn group, this group showed lower values for these variables than the spring group. Results show that at least in Iberian deer, counter-season reproduction is possible, and that the effects on lactation variables are similar to those of a natural delay in calving.     

Characteristics of the oestrous cycle of Iberian red deer (Cervus elaphus hispanicus) assessed by progesterone profiles

This study examines the length of the oestrous cycle in 16 Iberian red deer females assessed by means of changes in progesterone concentrations, along with the changes in the profile of this hormone. Samples were collected three occasions per week from the week after calving (15 May to 15 June) up to May of the following year. The oestrous cycle lasted 19.57+/-0.29 days (range 10-27 d) calculated in 130 oestrous cycles examined. Progesterone titres did not rise above 0.5 ng/ml in the follicular phase, except in four samples. The maximum peak in progesterone concentration during the luteal phase remained above 1 ng/ml in most cases. Twenty-five percent of the individuals studied (4 out of 16) showed an oestrous cycle lasting shorter than the mean (15.2+/-0.30 days) before the start of the reproductive season, followed by a period of sexual inactivity. The standard progesterone profile in natural oestrous cycles rose from basal levels to those above 0.5 ng/ml four days after onset of oestrus, reached a peak of 1.71+/-0.07 ng/ml and then declined to less than 0.2 ng/ml after day 20. Following the rapid decline of progesterone after day 14, the concentration remained around the baseline level of 0.1 to 0.2 ng/ml during the immediate pre- and post-ovulatory phase of the cycle.     

Effect of storage temperature during transport of ovaries on in vitro embryo production in Iberian red deer (Cervus elaphus hispanicus)

The aim of this work was to study the effect of storage temperature during the transport of ovaries on cleavage and blastocyst rates in Iberian red deer, because wild populations of this subspecies are usually far from laboratories. A total of 472 ovaries from 236 Iberian hinds were recovered and maintained in saline solution at 5-8 °C or 20-25 °C for 12 h. After storage, aspirated oocytes were matured with FSH/LH or EGF and the developed embryos were cultured with oviduct epithelial cells monolayer (OCM). A higher (P = 0.009) cleavage rate was obtained when the ovaries were stored at 5-8 °C. However, there were no differences between both storage temperatures in relation to the percentage of blastocysts obtained. Considering the management and production systems of Iberian red deer, this study provides important information about the ovary storage temperature during transport with the purpose of assuring an optimal in vitro embryo production.     

Milk intake and production curves and allosuckling in captive Iberian red deer, Cervus elaphus hispanicus

In two experiments, we compared milk intake (assessed by weighing calves before and after suckling) and milk production (by hand milking hinds) in Iberian red deer both when calves sucked their mothers together (group-suckling experiment) and when mother and offspring were isolated (isolation-suckling experiment). In both experiments, the general lactation curve for calves increased to a peak and then decreased (type I, standard lactation curve in mammals), whereas the curve for hinds decreased from the start (type II). However, in the experiment on group suckling, calves ingested 17.2% more milk than that produced by their mothers from weeks 6 to 20. In both isolation- and group-suckling experiments, hinds showed an overproduction of milk decreasing from weeks 1 to 5. This decreasing overproduction coincides with a similar trend in calf mortality reported in the literature and might thus be aimed at ensuring calves have sufficient nutrients when mortality is highest. In addition, allosuckling observations in the group-suckling experiment showed an inverse relationship between milk production and percentage of allosucking attempts. Allosucking attempts were also more frequent after the milk overproduction period. Both findings suggest that allosuckling is a response to compensate for a reduced maternal milk supply. Copyright 2000 The Association for the Study of Animal Behaviour.     

Effects of nutritional stress during lactation on immunity costs and indices of future reproduction in Iberian red deer (Cervus elaphus hispanicus)

Nutrition may affect the balance between immunity and traits such as reproduction or growth. This study examines the effect of low nutrient availability on immunity indices and lactation in captive Iberian red deer. Twelve hinds and their calves were allotted to a food-restricted (50-60% daily energy requirements) or a control group just after calving. Low calorie intake exerted a greater effect on the immunity of calves than on that of hinds. Whereas no difference was found for hinds, calves of the low intake group showed mean immunoglobulin (Ig) levels higher than those on a standard diet, which suggests that Ig level may indicate the level of fighting against pathogens. Serum indices of body condition in calves showed generally positive correlations with milk nutrient production. In contrast, Ig level within each group showed a pattern inverse to that of the other group for early lactation: in the standard diet group, the greater the milk nutrient produced and calf growth, the lower the Ig level; this relationship was inversed in the low-nutrition group. These results suggest that, on a standard diet, high Ig levels may indicate high levels of pathogen fighting paired to poorer body condition. Inversely, once the first barriers of innate immunity are surpassed, only those calves on the low-nutrition group with greater resources would be able to spend more resources to fight infection. Thus, low calorie intake might boost its slowing effect on growth by increasing the costs of infection fighting.     

Age-related body weight constraints on prenatal and milk provisioning in Iberian red deer (Cervus elaphus hispanicus) affect allocation of maternal resources

Maternal phenotypic characteristics can influence key life history variables of their offspring through maternal effects. In this study, we examined how body size constraints on maternal weight in yearling and subadult compared to adult hinds (age class effects) affected prenatal (calf birth weight, calf to hind weight ratio) and postnatal (milk) provisioning of Iberian red deer calves. Age correlated with all prenatal and postnatal investment traits except calf gains, although correlations were weaker than those with maternal weight. Once the effect of linear increase in weight with age was removed from models, yearlings showed additional reductions in calf birth weight, calf gains, and milk provisioning. The low-calf birth weight might increase the risk of calf mortality during lactation, as this occurs primarily during the first day of life and is strongly related to birth weight. Yearlings showed a greater prenatal allocation of resources in terms of greater calf to hind weight ratio probably as an extra effort by yearling mothers to balance calf neonatal mortality. It might compensate young mothers to produce low-quality calves while still growing rather than waiting for the uncertain possibility of surviving to the next reproductive season.     

First report of Elaphostrongylus cervi in Spanish red deer Cervus elaphus hispanicus

Elaphostrongylus cervi Cameron, 1931 was identified in six Cervus elaphus hispanicus sampled in Cuenca, central Spain. A total of 23 adult worms were found in the central nervous system with a mean of 3.8. Although E. cervi is reported to be widespread in cervids, this is the first time it has been recorded in Spanish red deer.     

Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate-fructose) osmolality (300-600 mOsm/kg), and (2) effect of sugar (0.4M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2h at 37 degrees C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious (P<0.05). In experiment 2, fructose had a positive effect respect to control after 2h of incubation at 37 degrees C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.     

Effect of melatonin implants on the incidence and timing of puberty in female red deer (Cervus elaphus)

A study was conducted to test the hypotheses that exogenous melatonin treatment of 11–13 month-old red deer hinds: (1) advances the timing of first ovulation, (2) increases the proportion of individuals attaining puberty at ～16 months of age, and (3) reduces the live-weight threshold for attainment of first pregnancy. A total of 3901 rising-2-year-old (R₂) hinds within two herds (A and B) across two years either received single melatonin implants on two occasions in summer (n = 1399) or were untreated controls (n = 2502). Hinds were joined with stags from mid January to mid May, and were subjected to real-time rectal ultrasonography in early June to assess pregnancy status (proxy for puberty attainment) and foetal age for conception date assignment. Live-weights were recorded for each hind in January (12 months of age) as a proxy for weight at puberty. Melatonin treatment of hinds was associated with a significant advancement in mean conception dates in both herds in both years (P < 0.05), with a cohort difference in mean dates between treated and control hinds ranging from 9 to 17 days. Analysis of the temporal distribution of conception dates for each cohort revealed bi-modal or tri-modal patterns of conception indicative of conceptions to first or subsequent ovulations (oestrous cycles). Across all cohorts, melatonin treatment was associated with higher conception rates to first ovulation (P < 0.05) resulting in greater overall synchrony of conceptions. Regression analysis demonstrated a significant negative slope for conception date against live-weight (P < 0.001), but there was no evidence that this slope varied with treatment, herd or year (P > 0.05); for every 10 kg increase in live-weight conception date was advanced by an average of 1.3 days. In Herd A, melatonin treatment was associated with significantly higher pregnancy rates in both years (90.3% vs. 78.0% in Year 1 and 84.4% vs. 57.1% in Year 2; P < 0.05). The principle effect of melatonin treatment was to increase the pregnancy rate of hinds of low body-mass. In Year 1, at 60 kg live-weight a logit regression model indicated a pregnancy rate of 52% for untreated hinds and 83% for treated hinds. At 105 kg the rate for both cohorts was 90%. In Herd B, melatonin treatment was associated with higher conception rates in both years but these differences were not significant following correction for slight differences in mean live-weight (P > 0.05). The study has demonstrated that factors influencing puberty attainment in R₂ red deer hinds can vary between populations. In Herd A, in which body mass of hinds immediately prior to their first potential breeding season may have been the principle limiting factor, melatonin treatment appears to have instigated the pubertal process in hinds that would otherwise be of insufficient body mass.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.     

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.