Season effect on genitalia and epididymal sperm from Iberian red deer, roe deer and Cantabrian chamois

Seasonality deeply affects the physiology and behavior of many species, and must be taken into account when biological resource banks (BRBs) are established. We have studied the effect of seasonality on many reproductive parameters of free-ranging Iberian red deer, roe deer and Cantabrian chamois, living in Spain. Testicles from hunted animals were collected and sent to our laboratory at different times during the year. We recorded the weight and volume of testis, the weight of the epididymis and its separate parts (caput, corpus, and cauda), the weight of the sperm sample collected from the cauda epididymis, and several sperm parameters (sperm concentration, spermatozoa recovered, motility, HOS test reactivity, acrosomal status, and viability). We studied the data according to several periods, defined accordingly to each species. For red deer, we defined rut (mid-September to mid-October), post-rut (mid-October to mid-December), and non-breeding season (February). For roe deer, they were pre-rut (June), rut (July), post-rut (first fortnight of August), and non-breeding season (September). For chamois: non-breeding season (June to mid-September) and breeding season (October-November). The rut/breeding season yielded significantly higher numbers for almost all parameters. However, in the case of red deer, sperm quality was higher in the post-rut. For roe deer, testicular weight was similar in the pre-rut and in the rut, and sperm quality did not differ significantly between these two periods, although we noticed higher values in the rut. In the case of chamois, sperm quality did not differ significantly from the breeding season, but data distribution suggested that in the non-breeding season there are less males with sperm of good quality. On the whole, we find these results of interest for BRB planning. The best season to collect sperm in this species would be the breeding season. However, post-rut in red deer, pre-rut in roe deer, and non-breeding season in chamois could be used too, because of the acceptable sperm quality, despite the lower quantity salvaged. More in-depth research needs to be carried out on the quality of sperm salvaged at different times of the year in order to confirm these findings.     

Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.     

Sperm parameters on Iberian red deer: electroejaculation and post-mortem collection

Artificial reproductive technologies (ART) for cervids have improved, but a need remains for the collection of basic data. We studied two models of sperm collection in Iberian red deer, post-mortem (PM) in a wild population (179 samples) and by electroejaculation (EE) in a farmed population (37 samples), recording: testicular and epididymal weight, testicular diameter, sperm quantity, pH and osmolality and spermatozoa quality (motility by CASA, abnormal forms, cytoplasmic droplets, viability and acrosomal status). We tested the relationship of these parameters with stag age and compared the two models (PM and EE; medians showed). Genitalia parameters were linearly related to stag age (testicular diameter: 31.5-50.5mm for 2-9 years). Total number of spermatozoa collected were PM: 2.5x10(9) and EE: 3.6x10(9) (P>0.05), increasing with age only for PM. We found a positive relationship between testicular size and spermatozoa collected for PM. Osmolality and pH were PM: 6.28 and 378mOsm/kg; EE: 7.63 and 309mOsm/kg (P<0.05). The pH increased with age only for EE. Percentage of motile spermatozoa was similar for PM and EE, but motility quality was lower for PM. Abnormal forms, proximal and distal droplets were lower for EE (22%, 1.3%, 1.5% vs. PM: 23%, 4.3%, 83%). Viability was similar (74%) and intact acrosomes were higher for EE (97% vs. 89%). Both PM and EE samples could be used for germplasm banking. This study contributes with new data on red deer spermatology and for the development of ART in cervids.     

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.     

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 season on characteristics of red deer /Cervus elaphus L./ semen collected using modified artificial vagina

During three reproductive seasons, 572 ejaculates were collected from five farmed red deer stags using a modified artificial vagina. The ejaculates were evaluated in terms of their quality and quantity by standard procedures applied for domestic animals. The total length of sexual activity was 245 days, from August 4 to April 6. Significant changes in the semen parameters were noted over this period, divided into three stages: pre-mating, mating, post-mating. During mating season the following values of sperm parameters were recorded: 1/ volume of white and yellow fractions -0.18 and 2.03 ml, respectively; 2/ pH of white and yellow fraction -6.80-7.41 and 6.65-7.45, respectively; 3/ sperm concentration -2.27 mln/mm3; 4/ sperm motility -47% (with 80% of them showing steady progressive motion at moderate speed); 5/ duration of sperm motility -6 hours 39 minutes, and 6/ low percentage of major and minor sperm defects (<5% and <10%, respectively). In summary, measures of semen quality including fraction volume, pH, sperm concentration and sperm motility change gradually during the pre-mating, mating and post-mating seasons of red deer. The period of greatest libido (from the end of September until the end October) coincides with highest semen quality.     

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.     

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility,viability, and morphological integrity

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.     

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.     

Assessment of in vitro fertility of deer spermatozoa by heterologous IVF with zona-free bovine oocytes

We have previously shown that the in vitro development of deer embryos differed according to the IVF conditions. The aim of the study was to use heterologous IVF with zona-free matured bovine oocytes to assess the in vitro fertility of 3 samples of deer semen (2 ejaculates from sika deer (Cervus nippon) and 1 pool of epididymal spermatozoa from red deer (Cervus elaphus)). The frozen/thawed semen samples were selected on Percoll gradient and resuspended in Tyrode modified medium supplemented with estrus sheep serum (0, 2, 20% v/v) or heparin (10 microg/mL). During 8 h of culture, the sperm motility index according to the post-insemination time (hpi) did not differ either between samples or between supplemented IVF media. In vitro matured zona-free bovine oocytes were inseminated in different IVF media with the semen samples. Penetration rates assessed at 15 hpi were optimal with 20% estrus sheep serum for sika deer ejaculates whereas 2% were sufficient to reach the maximum functionality of epididymal spermatozoa from red deer. The mean time of pronuclear formation was similar regardless of the semen sample. The precocity of the onset of the first S-phase in both pronuclei was characterized by Bromo-deoxy-Uridine exposures between 5 and 15 hpi in order to assess the developmental potential conferred by the semen sample (intrinsic value). As we previously observed in homologous IVF, this value seemed to be higher for the epididymal sperm sample.     

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.     

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.     

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.     

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).     

Effects of pH, lactate, and viscoelastic drag on sperm motility: a species comparison

Little or no motility is observed when sperm from 5 mammalian species are incubated in vitro in their cauda epididymal fluid (CEF). We examined the effects of pH, lactate, and viscoelastic drag on sperm motility to determine whether these factors are responsible for this inhibition of motility. The pHs of CEF from bull, dog, rat, guinea pig, and hamster were 5.8, 6.2, 6.9, 6.9, and 7.2, respectively. The lactate concentration of epididymal semen collected from anesthetized animals ranged from 0.6 to 0.9, but increased almost 10-fold in samples from rats or dogs when measured 2 h postmortem. Increasing the pH of CEF to 7.0 resulted in the initiation of full motility for bull and dog sperm. Suspensions of sperm in buffer at various pHs (from 4.0 to 7.6) produced a sigmoidal motility curve for all species. All species, including bull and dog, showed almost full motility in buffer at a pH equal to the pH of their own CEF. Motility of bull and dog sperm showed greater inhibition with decreasing pH when suspended in CEF instead of buffer. The addition of 15 mM lactate, which has been shown to lower sperm intracellular pH, shifted the motility versus pH curves of all species toward higher pH. In bull and dog the addition of lactate produced a motility profile that was indistinguishable from that in their own CEF. The viscoelastic drag of the CEF of only two species, rat and hamster, was sufficiently high to inhibit sperm motility. We conclude that the low pH of the CEF from bulls and dogs plus the presence of lactate is sufficient to cause inhibition of motility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 × g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4 h was assessed using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30 ± 3.52% to 67.94 ± 5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.     

Horn quality and postmortem sperm parameters in Spanish ibex (Capra pyrenaica hispanica)

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility.     

Influence of livestock,habitat type,and density of roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>) on parasitic larvae abundance and infection seroprevalence in wild populations of roe deer from central Iberian Peninsula

Livestock farming is a common human activity that not only modifies natural habitat but also may lead to interactions with other wild animal species. We studied whether health status of roe deer (Capreolus capreolus) is influenced by density, livestock, and type of habitat. We analyzed 208 samples (120 fecal and 88 sera) from roe deer populations of central Iberian Peninsula to evaluate both presence and abundance of parasitic oocysts, eggs, and/or larvae, especially of gastrointestinal and bronchopulmonary parasites, as well as the prevalence of five infectious diseases (infectious bovine rhinotracheitis (IBR), pestivirosis (BVD/BD), paratuberculosis (PTB), bluetongue (BT), and brucellosis). Fecal samples were collected in transects in pine and oak forests and endoparasites were detected by means of coprological techniques. Serum samples were obtained from hunted individuals and the serological testing for the infectious pathogens was performed by ELISA and Rose Bengal tests. Livestock and habitat type were associated both with the presence and the number of bronchopulmonary nematode L1-larvae per gram of feces (lpg), which was higher in areas with livestock and in pine forests. However, roe deer density neither influenced parasite presence nor lpg values. Regarding infectious pathogens, only seropositive samples for PTB and pestivirus were obtained indicating a possible transmission between domestic and wild ungulates in the study area. The low prevalence found is consistent with other studies in the Iberian Peninsula suggesting that roe deer have little contact with the infectious agents studied. Our results highlight that both habitat type and livestock significantly mediate abundance of parasitic larvae in roe deer, being higher under competition scenarios and in habitats of lower quality, a valuable aspect to be considered in future management and conservation strategies.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P<0.001). Penetration rates were similar for 4-12h pre-incubation of sperm on COEM (P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study.It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.