Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential

Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males.After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure.Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations.     

Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats

The aim of this study was to determine if there is a seasonal pattern of reproductive activity in male Payoya goats and if this seasonality can be modulated by a higher level of nutrition. For a period of 16 months, 10 adult bucks were divided into two experimental groups that differed in their feeding level. The high nutrition group (H, n = 5) received 1.6 times their maintenance food requirements. The control nutrition group (C, n = 5) received a diet that supported 1.1 times their maintenance requirements. Body weight and testosterone concentrations were determined weekly, and testicular weight was determined every 2 weeks. Sexual behaviour and semen characteristics were determined monthly. Feeding level did not affect the onset or the end of the reproductive activity as measured by testosterone concentrations, with high testosterone concentrations between July and November. Ejaculation latency was positively influenced by feeding level: 43.2 ± 2.2 s vs. 61.6 ± 3.2 s for H and C group, respectively (P < 0.001). The percentage of males that ejaculated or that were sexually active was higher in the H group (P < 0.01). No differences between feeding levels were observed in the different semen characteristics studied. However, major differences between months were observed for all studied variables. These results lead us to conclude that Payoya bucks exhibit large seasonal variation in their reproductive activity. Higher feeding level allowed a better sexual behaviour in bucks in late spring, when male effect is used on the local livestock to breed females.     

Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I

The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo (Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17 ± 1.51) compared with that in the control group (9.50 ± 0.36) at 2 h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30 min (33.27 ± 2.62 vs. 26.71 ± 1.02), 60 min (24.24 ± 3.45 vs. 18.77 ± 2.09), and 90 min (22.86 ± 3.02 vs. 16.92 ± 1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120 min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2 h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2 h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4 h (41.12 ± 6.44 vs. 43.53 ± 5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I-treated group (56.73 ± 3.70) compared with that in the control group (44.85 ± 2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility.     

The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine (Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

Pregnancy-associated changes in plasma concentration of the endocrine disruptor di(2-ethylhexyl) phthalate in a sheep model

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45 ± 0.01 μg/mL (range, 0.05 to 2.81 μg/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n = 66, 37.9%) than in younger animals (n = 69, 17.4%; P < 0.05), although the mean levels in ewes with residues were similar (0.16 ± 0.01 vs. 0.16 ± 0.02 μg/mL). All the pregnant ewes (n = 15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42 ± 0.18 vs. 0.16 ± 0.01 μg/mL; P < 0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n = 32, 71.9%; P < 0.005) than in nonpregnant ewes (n = 37, 35.1%) or in ewes that recently gave birth (n = 32, 21.9%), although mean levels were similar (0.42 ± 0.02, 0.33 ± 0.02, and 0.34 ± 0.05 μg/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

Semen characteristics of genetically identical male cats cloned via somatic cell nucleus transfer

We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (× 10⁶/mL) of Cat A (512 ± 140, range 368 to 685) was significantly higher, whereas that of Cat C (335 ± 92, range 274 to 469) was significantly lower than that of Cloned B (459 ± 159, range 336 to 510) and control cats (680 ± 452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage = 74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats.     

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus)

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean ± SEM: 0.90 ± 0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95 ± 1.73 μg/dL) was significantly higher in April. Average number of spermatozoa was 10.0 ± 3.4 × 10⁶ sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7 ± 5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7 ± 2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7 ± 3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3 h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.     

Effect of transgenesis on reproductive traits of rabbit males

The influence of foreign transgene integration on the reproductive capabilities of rabbit males is not known. Therefore, we analyzed their ejaculate characteristics, reproductive capabilities, occurrence of pathological spermatozoa and histological structure of the testis. We have generated transgenic rabbits by microinjection of WAP-hFVIII gene into pronucleus of fertilized egg. We observed that the libido, volume and pH value of the ejaculate did not differ significantly between transgenic and non-transgenic male lines. The motility, concentration, osmolarity, thermoresistant test of spermatozoa (at 1 or 6 h) and the percentage of alive spermatozoa were significantly different (p < 0.001) among transgenic and non-transgenic males. No significant differences were found between transgenic and non-transgenic male lines in the occurrence of pathological spermatozoa and histology of the testis. The ability of spermatozoa from transgenic and non-transgenic males to fertilize eggs was ranged within 96 and 100%; while the yield of transgenic embryos ranged from 43 to 57%. Our results show that mammary gland specific over-expression mWAP-hFVIII gene construct does not affect reproductive traits of transgenic rabbit males.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Influence of different reproductive rhythms on serum estradiol and testosterone levels,features of follicular population and atresia rate,and oocyte maturation in controlled suckling rabbits

The aim of the current work was to analyze the features of ovarian follicular population and their quality in New Zealand white rabbit does synchronized by 24-h controlled doe-litter separation before artificial insemination (AI) during all their reproductive cycles. Synchronized animals were allocated systematically in two groups. A total of 73 rabbit does (group A) were submitted to a 35-day intensive rhythm (AI on day 4 post-partum [pp] and weaning at 25 days of lactation), and 108 rabbit does (group B) were submitted to a 42-day semi-intensive rhythm (AI on day 11 pp and weaning at 35 days of lactation) during 9 months. At the mid-end of their reproductive life, a total of 26 does (5.4 parturitions), under intensive (n = 15) or semi-intensive rhythm (n = 11) were either treated in each group with 25 IU eCG 48 h before laparotomy to recover their ovaries (n = 7 for group A and n = 6 for group B) (according to the Bioethics Committee of the University) or not synchronized with the hormonal treatment (n = 8 for group A and n = 5 for group B). Blood samples were collected at the moment of ovary recovery; morphometrical parameters, number of total follicles and number of follicles ≥1 mm in size in the ovarian surface were recorded. Oocytes from follicles of one ovary were recovered and matured in TCM 199 supplemented with 10 ng/ml EGF, 100 ng/ml IGF-I and 10% FCS. The counterpart ovaries were fixed in paraformaldehyde solution for histological studies. Detection of cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) technique. Reproductive performance was affected by the rhythm used, with lower reproductive parameters in the intensive group. The average ovary height and width, the mean number of ≥1 mm follicles and the number of total follicles were similar between groups. Serum concentrations of estradiol (E₂) and testosterone (T) were significantly lower in group A vs. B (E₂: 232.4 ± 56.1 vs. 399.7 ± 53.0 pg/ml; P < 0.05 and T: 1.07 ± 0.10 vs. 1.68 ± 0.23 ng/ml; P < 0.05). No significant differences were found in follicular population or in the mean follicular apoptosis index between groups. Metaphase II rate was significantly lower in group A vs. B (48.5 ± 3.3 vs. 67.6 ± 3.7%; P < 0.01), as well as the migration rate of cortical granules (12.7 ± 2.7 vs. 38.2 ± 6.6%; P < 0.001). On the other hand, neither follicular population, nuclear maturation rate nor apoptosis rate were affected by the eCG treatment, but cytoplasmic maturation was higher in animals treated with eCG in group A (29.2% vs. 5.5%; P < 0.01). In conclusion, rabbit does under transient litter separation during their reproductive life have both their serum estradiol and testosterone concentrations and oocyte quality influenced by the intensive rhythm, leading to a decrease in reproductive parameters. Also, both intensive and semi-intensive rhythms seem to be less receptive to eCG treatment than expected.     

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24 h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24 h post activation.A forward progressive movement was maintained for at least a 20 h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6 h post activation and severe alterations were observed in sperm morphology after 24 h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.     

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P = 0.0201) in BoHV-FBS (80.4 ± 8.9%; mean ± SD) compared with that of noninfected oocytes (89.9 ± 6.5%); neither differed from BoHV-PVA (87.3 ± 7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5 ± 7.5% vs. 92.2 ± 5.5%, control vs. infected) and development of embryos (41.7 ± 9.9% vs. 44.3 ± 7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6 ± 10.3% vs. 40.8 ± 9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P = 0.1462, P = 0.5402, and P = 0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P = 0.0140 to M/B/EB and P = 0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6 ± 4.6 vs. 25.0 ± 5.5 and 31.6 ± 4.6 vs. 20.2 ± 5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.     

Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

Características de la fractura de cadera y posterior recuperación en pacientes mayores de 65 años con historia de caídas recurrentes

Recurrent falls affect between 14.8% and 19% of the elderly population, and are associated with an increased risk of fracture. We know little about the influence the history of recurrent falls may have on recovery after hip fracture.

Methods

Cohort study. The patients included were, over 65 years admitted during a 1 year period to the General University Hospital of Albacete with a hip fracture due to a fall. Recurrent falls were defined as a history of two or more falls within the 6 months prior to the fracture. Variables: demographic data, circumstances of fall, number of falls in the previous 6 months, type of fracture and its repair, comorbidity and drug treatment, cognitive status at admission (Pfeiffer test) and independence for activities of daily living (Barthel Index - BI) were collected. A subsample of patients with pre-fracture BI≥60 and Pfeiffer at admission≤4 was followed up at 3, 6 and 12 months.

Results

A total of 335 patients were admitted. Data were collected on 279 of them, 19.4% of whom had previously suffered two or more falls. The recurrent fallers had a worse mental status on admission, a higher number of associated diseases, a lower percentage of independence in dressing and in bed-chair transferring than patients without history of recurrent falls, all statistically significant. In the 201 patients followed up, the impairment on the BI after 12 months compared to the BI previous to fracture was higher in recurrent fallers (-20.8 ± 31.54 vs -10.73 ± 20.21, P = .04), focusing more on independence in eating (76% vs 91.9%, P < .05), grooming (72% vs 91,9%, P < .01), faecal continence (60% vs 78.7%, p < .05) and walking indoors (80% vs 93.3%, P < .05).

Conclusions

The recovery of independence after hip fracture is significantly lower in the group of recurrent fallers in patients without moderate or severe functional impairment previous to fracture and cognitively stable.     

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.     

In vitro effects of bisphenol A on the quality parameters,oxidative stress,DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10 μg/L) for 2 h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5–10 μg/L. Significant positive correlation (r = 0.713, P < 0.05) was found between percent motile spermatozoa and ATP content. Oxidative stress was observed at concentrations 1.75–10 μg/L, as reflected by significantly higher levels of protein and lipid oxidation and superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of BPA. A dramatic increase in DNA fragmentation expressed as percent tail DNA (2.2% ± 0.46) and Olive tail moment (0.37 ± 0.09 arbitrary units) was recorded at concentrations of 1.75 μg/L and above. The present study confirms that concentrations of BPA that can be encountered in nature are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content.     

Seasonality of reproduction in wild boar (Sus scrofa) assessed by fecal and plasmatic steroids

The collection of biological samples through non-invasive techniques represents one way of monitoring in vivo physiological changes associated with reproductive activity. Such techniques are particularly important for the study of animal species in the wild.The goals of this study were 1) to evaluate fecal progestogen (P), estrogen (E), and androgen (A) by means of radioimmunoassays, in male and female wild boars culled in the Piedmont, Italy area; 2) to compare them with plasmatic concentrations and the animals’ reproductive status; and 3) to assess variations in reproductive seasonality between two populations of wild boars living in a mountainous vs. a plain habitat in Piedmont.The results demonstrate a positive correlation between fecal and plasmatic steroid concentrations (r = 0.46, 0.58, and 0.45 for plasma P₄ and P, E₂ and E, and T and A; P < 0.05). Moreover, high fecal levels of both P and E (>170 ng/g and >100 pg/g respectively) were found in 70.6% of pregnant sows and in none of the non-pregnant animals, thus supporting the use of this technique for detecting pregnancy status in wild boar.Similar birth patterns were displayed by the mountain and plain populations, but births peaked significantly only in the mountain population, in the spring (46%, P < 0.05, vs. other seasons). A corresponding autumnal peak of plasma testosterone concentrations in males was displayed only by the mountain population (7.4 vs. < 2.0 ng/mL in the other seasons, P < 0.05).The correlation between fecal and plasmatic steroid concentrations obtained in this study supports the applicability of this non-invasive sampling technique for monitoring reproductive status in wild boar, thus enabling a more informed and correct management of the species.