The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

兔球虫与大肠杆菌病混合感染的诊治

阐述了兔球虫与大肠杆菌病混合感染的临床症状、实验室检验及防治方法,力求为科学防控两病提供有益的参考。     

Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance for Sperm Functionality In Vitro

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.     

Prevalence of dental and mandibular disorders in culled sheep in Spain

At the slaughterhouse, 1465 culled sheep heads were studied in order to identify disorders of the mandibular cheek teeth. Of these, 227 (15.5%) had evidence of osteomyelitis. The lesional profile showed that the mandible was affected in a similar proportion on both sides (46.7% on the right side vs 50.7% on the left side), mainly in the middle region (55.3%) and with most of the lesions closed without fistulisation (89.4%). In addition, swelling was palpable, with an increase in thickness in the area of the affected body (2.65 ± 0.065 cm vs 1.74 ± 0.030 cm). In 78.6% of the animals, the regional lymph nodes were enlarged. Concerning the mandibular cheek teeth, more than half of the animals lacked at least one tooth (57.0%), with the first premolar being the most frequently missing tooth (34.8%) and the third molar the least (8.2%). The impaction of food around the teeth was very common with the posterior molars being more frequently affected. In the knowledge of the authors, this is the first study that analyses the prevalence of mandibular molar and premolar disorders in sheep, and these are revealed as an important condition affecting culled sheep.     

Serial Transrectal Ultrasonography for Monitoring the Reproductive Activity of the Asiatic Black Bear (Ursus thibetanus ussuricus)

This study evaluated the structural changes in the reproductive tract of Asiatic black bears using serial transrectal ultrasonography. In addition, the ultrasonographic observations were compared with the results of vaginal cytology and hormonal analyses. The collection of blood for hormonal analysis, vaginal cytology and transrectal ultrasonography was performed in two bears (Bears 1 and 2) from June 2011 to August 2013 without mating and in a third bear (Bear 3) from April to December 2012, allowing natural mating. Serial ultrasonographic observations showed cyclic changes in ovarian structures (e.g. emergence of small follicles, growth and ovulation of dominant follicles and corpus luteum (CL) formation) during the reproductive cycles of the three bears. The diameter of the uterine horns remained similar throughout the reproductive cycle in Bears 1 and 2, and it remained similar from April until October, but an enlargement containing foetuses was observed in Bear 3 in December. The ultrasonographic observations were consistent with the data obtained through vaginal cytology and progesterone analysis during the reproductive cycle. An average of 4.0 (±0.4) dominant follicles was observed during the oestrous stage (May‐August), during which the superficial cells accounted for >90% of the total vaginal cells. In addition, the detection of an average of 2.6 (±0.2) CL was associated with increased plasma progesterone concentrations (3.0 ± 0.4 ng/ml) between June and December (near hibernation). In conclusion, serial transrectal ultrasonography demonstrated yearly oestrous (ovulation) cycles via follicular dynamics and CL formation on ovaries, accordingly with vaginal cytology and hormonal level in the Asiatic black bear.     

Ancient biomolecules from deep ice cores reveal a forested southern Greenland

It is difficult to obtain fossil data from the 10% of Earth's terrestrial surface that is covered by thick glaciers and ice sheets, and hence, knowledge of the paleoenvironments of these regions has remained limited. We show that DNA and amino acids from buried organisms can be recovered from the basal sections of deep ice cores, enabling reconstructions of past flora and fauna. We show that high-altitude southern Greenland, currently lying below more than 2 kilometers of ice, was inhabited by a diverse array of conifer trees and insects within the past million years. The results provide direct evidence in support of a forested southern Greenland and suggest that many deep ice cores may contain genetic records of paleoenvironments in their basal sections.     

Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection

Background

Parasites that manipulate host behavior can provide prominent examples of extended phenotypes: parasite genomes controlling host behavior. Here we focus on one of the most dramatic examples of behavioral manipulation, the death grip of ants infected by Ophiocordyceps fungi. We studied the interaction between O. unilateralis s.l. and its host ant Camponotus leonardi in a Thai rainforest, where infected ants descend from their canopy nests down to understory vegetation to bite into abaxial leaf veins before dying. Host mortality is concentrated in patches (graveyards) where ants die on sapling leaves ca. 25 cm above the soil surface where conditions for parasite development are optimal. Here we address whether the sequence of ant behaviors leading to the final death grip can also be interpreted as parasite adaptations and describe some of the morphological changes inside the heads of infected workers that mediate the expression of the death grip phenotype.

Results

We found that infected ants behave as zombies and display predictable stereotypical behaviors of random rather than directional walking, and of repeated convulsions that make them fall down and thus precludes returning to the canopy. Transitions from erratic wandering to death grips on a leaf vein were abrupt and synchronized around solar noon. We show that the mandibles of ants penetrate deeply into vein tissue and that this is accompanied by extensive atrophy of the mandibular muscles. This lock-jaw means the ant will remain attached to the leaf after death. We further present histological data to show that a high density of single celled stages of the parasite within the head capsule of dying ants are likely to be responsible for this muscular atrophy.

Conclusions

Extended phenotypes in ants induced by fungal infections are a complex example of behavioral manipulation requiring coordinated changes of host behavior and morphology. Future work should address the genetic basis of such extended phenotypes.     

Quantifying Transport Between the Tropical and Mid-Latitude Lower Stratosphere

Airborne in situ observations of molecules with a wide range of lifetimes (methane, nitrous oxide, reactive nitrogen, ozone, chlorinated halocarbons, and halon-1211), used in a tropical tracer model, show that mid-latitude air is entrained into the tropical lower stratosphere within about 13.5 months; transport is faster in the reverse direction. Because exchange with the tropics is slower than global photochemical models generally assume, ozone at mid-latitudes appears to be more sensitive to elevated levels of industrial chlorine than is currently predicted. Nevertheless, about 45 percent of air in the tropical ascent region at 21 kilometers is of mid-latitude origin, implying that emissions from supersonic aircraft could reach the middle stratosphere.     

Treatment of Unobserved Oestrus in a Dairy Cattle Herd with Low Oestrous Detection Rate up to 60 days Post-partum

The efficiency of treatments for unobserved oestrus and their effect on the reproductive performance of a dairy cattle herd with low oestrous detection rate till 60 days post‐partum (dpp), attributed to the declivous and slippery concrete floor were investigated. The herdsman requested advice in order to improve the mean days open of the herd, but no investments were allowed because a new unit was about to be built. Due to the low oestrus detection rate of the herd, the breeding policy was to inseminate at the first detected post‐partum oestrus. Cows were examined at 20–30 dpp to assess uterine involution, ovarian activity and prevalence of reproductive disorders and, at 60 dpp if no previous oestrus was detected. Each examination included palpation per rectum, ultrasound scanning and collection of a blood sample for plasma progesterone (P4) measurement. Cows with unobserved oestrus till 60 dpp were allocated either to a treatment group (n=139) or to a control group (n=139). Three treatments were used: (a) injection of PGF_2α (PG) upon detection of a corpus luteum (CL; n = 30), cows not observed in oestrus being re‐injected 11–12 days later. AI was at oestrus; (b) PRID (n=35) or Crestar (n=74) devices kept in situ for 12 and 9 days, respectively, were associated to an injection of PG and of equine chorionic gonadotrophin (eCG) at device removal. Cows were double‐fixed time‐inseminated at 48 and 72 h after device removal. All treated cows were examined at 48–72 h after treatment to confirm oestrus. The percentage of cows detected in oestrus up to 60 dpp remained unchanged through the trial (35 and 47% for years before intervention: 1994–95; 51 and 48% for years of intervention: 1996–97). In contrast, the oestrous detection rate was high both in treated (93%) and control (100%) cows. This possibly resulted from an improvement in the oestrous detection efficiency of the herd's personnel and from examination of cows at 48–72 h after treatment. Treated and control cows had identical conception rate (CR; 36 and 37%, respectively) and reproductive performance. However, the mean days open of the herd in 1996 was significantly improved in comparison with previous years (mean ± SEM: 134 ± 6, 126 ± 5, 110 ± 4 and 114 ± 5 days, for years 1994, 1995, 1996 and 1997, respectively, p < 0.05, ANOVA ). Conception rate to AI up to 40 dpp was significantly reduced, compared with the period between 60 and 100 dpp but, mean days open were significantly improved in cows inseminated up to 60 dpp, compared with thereafter (p < 0.05).