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Self-initiated sensory action effects are widely assumed to lead to less intense perception and reduced neural responses compared to externally triggered stimuli (sensory attenuation). However, it is unclear if sensory attenuation occurs in all cases of action-effect prediction. Specifically, when predicted action-effects are relevant to determine follow-up actions attenuation could be detrimental. We quantified auditory event-related potentials (ERP) in electroencephalography (EEG) when human participants created two-sound sequences by pressing two keys on a keyboard associated with different pitch, giving rise to identity-specific action-effect prediction after the first keypress. The first sound corresponded to (congruent) or violated (incongruent) the predicted pitch and was either relevant for the selection of the second keypress to correctly complete the sequence (Relevance) or irrelevant (Control Movement), or there was only one keypress and sound (Baseline). We found a diminished P2-timed ERP component in incongruent compared to congruent trials when the sound was relevant for the subsequent action. This effect of action-effect prediction was due to an ERP reduction for incongruent relevant sounds compared to incongruent irrelevant sounds at P2 latencies and correlated negatively with modulations of pupil dilation. Contrary to our expectation, we did not observe an N1 modulation by congruency in any condition. Attenuation of the N1 component seems absent for predicted identity-specific auditory action effects, while P2-timed ERPs as well as pupil size are sensitive to predictability, at least when action effects are relevant for the selection of the next action. Incongruent relevant stimuli thereby take a special place and seem to be subject to attentional modulations and error processing., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

In natural environments objects comprise multiple features from the same or different sensory modalities but it is not known how perception of an object is affected by the value associations of its constituent parts. The present study compares intra- and cross-modal value-driven effects on behavioral and electrophysiological correlates of perception. Human participants first learned the reward associations of visual and auditory cues. Subsequently, they performed a visual discrimination task in the presence of previously rewarded, task-irrelevant visual or auditory cues (intra- and cross-modal cues, respectively). During the conditioning phase, when reward associations were learned and reward cues were the target of the task, high value stimuli of both modalities enhanced the electrophysiological correlates of sensory processing in posterior electrodes. During the post-conditioning phase, when reward delivery was halted and previously rewarded stimuli were task-irrelevant, cross-modal value significantly enhanced the behavioral measures of visual sensitivity, whereas intra-modal value produced only an insignificant decrement. Analysis of the simultaneously recorded event-related potentials (ERPs) of posterior electrodes revealed similar findings. We found an early (90-120 ms) suppression of ERPs evoked by high-value, intra-modal stimuli. Cross-modal stimuli led to a later value-driven modulation, with an enhancement of response positivity for high- compared to low-value stimuli starting at the N1 window (180-250 ms) and extending to the P3 (300-600 ms) responses. These results indicate that sensory processing of a compound stimulus comprising a visual target and task-irrelevant visual or auditory cues is modulated by the reward value of both sensory modalities, but such modulations rely on distinct underlying mechanisms., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Vakhrushev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As important pollinators, honey bees play a crucial role in both maintaining the ecological balance and providing products for humans. Although several versions of the western honey bee genome have already been published, its transcriptome information still needs to be refined. In this study, PacBio single-molecule sequencing technology was used to sequence the full-length transcriptome of mixed samples from many developmental time points and tissues of A. mellifera queens, workers and drones. A total of 116,535 transcripts corresponding to 30,045 genes were obtained. Of these, 92,477 transcripts were annotated. Compared to the annotated genes and transcripts on the reference genome, 18,915 gene loci and 96,176 transcripts were newly identified. From these transcripts, 136,554 alternative splicing (AS) events, 23,376 alternative polyadenylation (APA) sites and 21,813 lncRNAs were detected. In addition, based on the full-length transcripts, we identified many differentially expressed transcripts (DETs) between queen, worker and drone. Our results provide a complete set of reference transcripts for A. mellifera that dramatically expand our understanding of the complexity and diversity of the honey bee transcriptome.

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zi Long Wang reports financial support was provided by National Natural Science Foundation (No. 32160134). Zi Long Wang reports financial support was provided by Natural Science Foundation of Jiangxi Province (No. 2018ACB21028)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A 5-year-old, clinically normal intact female Miniature Schnauzer was presented for demonstrative ultrasonography in a seminar. She had two pregnancies in the past and had a natural mating 2 months previously. Ultrasonography revealed a segmental and circumferential mural thickening of the right uterine horn. The endometrium was markedly thickened with multiple organized hyperechoic linear striations, perpendicular to the mucosal surface. Histology revealed focal endometrial hyperplasia resembling the maternal tissue of the normal canine placenta. A diagnosis of spontaneous pseudo-placentational endometrial hyperplasia (PEH) was made. This study described a unique ultrasonographic appearance of PEH, which may facilitate the diagnosis of PEH., (© 2020 National Marine Mammal Foundation. Veterinary Radiology & Ultrasound published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Radiology.)

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae., (© 2021 Royal Entomological Society.)

Reward value guides goal-directed behavior and modulates early sensory processing. Rewarding stimuli are often multisensory, but it is not known how reward value is combined across sensory modalities. Here we show that the integration of reward value critically depends on whether the distinct sensory inputs are perceived to emanate from the same multisensory object. We systematically manipulated the congruency in monetary reward values and the relative spatial positions of co-occurring auditory and visual stimuli that served as bimodal distractors during an oculomotor task performed by healthy human participants (male and female). The amount of interference induced by the distractors was used as an indicator of their perceptual salience. Our results across two experiments show that when reward value is linked to each modality separately, the value congruence between vision and audition determines the combined salience of the bimodal distractors. However, the reward value of vision wins over the value of audition if the two modalities are perceived to convey conflicting information regarding the spatial position of the bimodal distractors. These results show that in a task that highly relies on the processing of visual spatial information, the reward values from multiple sensory modalities are integrated with each other, each with their respective weights. This weighting depends on the strength of prior beliefs regarding a common source for incoming unisensory signals based on their congruency in reward value and perceived spatial alignment., (Copyright © 2020 Cheng et al.)

In the present study, we evaluated the utility of an intraluminal agarose stent (IAS) for end-to-end intestinal anastomoses in rabbits. Female New Zealand white rabbits (n = 14) underwent conventional sutured anastomosis (CSA) with or without an IAS. IAS were used to maintain the luminal diameter for more rapid and accurate suturing, and then was squeezed transluminally to crush it into fragments, which passed through the intestines and were eliminated. The rabbits were euthanized on postoperative day 21. At necropsy, the anastomoses were assessed for adhesion formation, stenosis, and bursting pressure and were examined histologically for collagen content and blood vessel formation. Anastamosis surgery took less time in the IAS group (15.0 ± 2.6 min) than in the CSA-only group (30.1 ± 7.9 min). Only 1 postoperative death occurred (in the CSA group), and postmortem examination revealed evidence of anastomotic leakage. Adhesion formation and stenosis did not differ between groups, but bursting pressure, collagen content, and blood vessel formation were all significantly increased in the IAS group. IAS may decrease the operative time by maintaining a clear surgical field at the anastomotic site. In addition, the use of IAS promotes rapid healing and maintains the luminal diameter during end-to-end intestinal anastomosis.

The current study aimed to investigate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen was collected from Duroc boars and was allocated to 32 treatment groups for cryopreservation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI) was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b) dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference observed among these three treatments. The farrowing rates after AI with post-thawed semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa., (Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Purpose: Musician's dystonia (MD) is a task-specific movement disorder related to extensive expert music performance training. Similar to other forms of focal dystonia, MD involves sensory deficits and abnormal patterns of sensorimotor integration. The present study investigated the impaired cortical sensorimotor network of pianists who suffer from MD by employing altered auditory and tactile feedback during scale playing with multichannel EEG., Methods: 9 healthy professional pianists and 9 professional pianists suffering from right hand MD participated in an experiment that required repeated scale playing on a MIDI piano under altered sensory feedback while EEG was measured., Results: The comparison of EEG data in healthy pianists and pianists suffering from MD revealed a higher degree of inter-regional phase synchronisation between the frontal and parietal regions and between the temporal and central regions in the patient group and in conditions that are relevant to the long-trained auditory-motor coupling (normal auditory feedback and complete deprivation of auditory feedback), but such abnormalities decreased in conditions with delayed auditory feedback and altered tactile feedback., Conclusions: These findings support the hypothesis that the impaired sensorimotor integration of MD patients is specific to the type of overtrained task that the patients were trained for and can be modified with altered sensory feedback.

Indoxyl sulfate (IS), a protein-bound uraemic toxin, has been found to accumulate in the serum of people with renal diseases and is associated with free radical induction, nephrotoxicity cardiovascular toxicity, and osteoblast cytotoxicity. Although IS has been studied in humans and in experimental models, the role of IS in dogs and cats with kidney disease has not been investigated. A high performance liquid chromatography system was applied to detect plasma IS concentrations in non-azotaemic animals (63 dogs, 16 cats) and in animals with renal azotaemia (66 dogs, 69 cats). The IS levels of azotaemic animals were significantly higher (P <0.01) than those of non-azotaemic animals (median [IQR] 20.4 (9.5) mg/L vs. 7.2 (8.8) mg/L for dogs; median [IQR] 21 (18.9) mg/L vs. 14.8 (12.3) mg/L for cats). The IS level was significantly correlated with blood urea nitrogen, serum creatinine and phosphate concentrations. Dogs with acute kidney injury had significantly higher IS levels (P <0.01) than those with chronic kidney diseases (CKD) (median [IQR] 57.7 (40.8) mg/L vs. 17.7 (25.1) mg/L). When CKD was graded using the International Renal Interest Society (IRIS) staging system, IS levels were correlated with CKD severity in both dogs and cats. The IS concentration is directly related to loss of renal function. Further studies are necessary to determine whether measurement of IS provides any additional diagnostic or prognostic information in dogs and cats with kidney disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP-2 and MMP-2 activity in high than low sperm concentration samples (p < 0.001). ProMMP-9 and MMP-9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP-2 and MMP-2 were associated with high sperm motility (≥70%, p < 0.001). Sperm-rich fraction showed significantly (eight-fold) higher proMMP-9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP-2-specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP-9, TIMP-1 and TIMP-2 were absent in these cells. Matrix metalloproteinase-9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP-1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP-2 localization along acrosomal region of sperm, while MMP-9, TIMP-1 and TIMP-2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP-2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP-2 and MMP-9 may serve as an alternative biomarker in determining semen quality., (© 2014 Blackwell Verlag GmbH.)

Background: This study investigates the effect of altered auditory feedback (AAF) in musician's dystonia (MD) and discusses whether AAF can be considered as a sensory trick in MD. Furthermore, the effect of AAF is compared with altered tactile feedback, which can serve as a sensory trick in several other forms of focal dystonia., Methods: The method is based on scale analysis (Jabusch et al., 2004). Experiment 1 employs synchronization paradigm: 12 MD patients and 25 healthy pianists had to repeatedly play C-major scales in synchrony with a metronome on a MIDI-piano with three auditory feedback conditions: (1) normal feedback; (2) no feedback; (3) constant delayed feedback. Experiment 2 employs synchronization-continuation paradigm: 12 MD patients and 12 healthy pianists had to repeatedly play C-major scales in two phases: first in synchrony with a metronome, secondly continue the established tempo without the metronome. There are four experimental conditions, among them three are the same AAF as in Experiment 1 and 1 is related to altered tactile sensory input. The coefficient of variation of inter-onset intervals of the key depressions was calculated to evaluate fine motor control., Results: In both experiments, the healthy controls and the patients behaved very similarly. There is no difference in the regularity of playing between the two groups under any condition, and neither did AAF nor did altered tactile feedback have a beneficial effect on patients' fine motor control., Conclusions: The results of the two experiments suggest that in the context of our experimental designs, AAF and altered tactile feedback play a minor role in motor coordination in patients with musicians' dystonia. We propose that altered auditory and tactile feedback do not serve as effective sensory tricks and may not temporarily reduce the symptoms of patients suffering from MD in this experimental context.

This study was to evaluate the combinatorial effect (14 treatments, A-N) of different Equex STM paste concentrations, cryoprotectants and the straw-freezing method on the post-thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post-thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post-thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw-freezing method changes between glycerol and DMA., (© 2012 Blackwell Verlag GmbH.)

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our laboratory recently confirmed the first case of encephalitozoonosis in a rabbit in Taiwan; the prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being infected. This study surveys the seropositivity of E. cuniculi using carbon immunoassay (CIA) and enzyme-linked immunosorbent assay (ELISA). Serological examination of 171 rabbits using CIA and ELISA showed that 63.2% (108/171) and 67.8% (116/171) were seropositive against E. cuniculi, respectively. Thirteen of the 14 rabbits (92.9%) with neurological symptoms were seropositive. Except for gender, health status and location had a significant effect on E. cuniculi seropositivity (p<0.05). Adult rabbits aged older than 4 months exhibited significantly higher seropositivity for E. cuniculi than young rabbits (p<0.05). In conclusion, this study shows that E. cuniculi is present and widespread among healthy rabbits in Taiwan. Therefore, the fields of veterinary and human medicine in Taiwan should be aware of this zoonotic issue and the resulting public health concern of encephalitozoonosis., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The renal resistive index (RI) value of 0.73 has been proposed as the upper limit in normal adult dogs. In humans, changes in RI with age are associated with plasma renin activity. There are relatively few equivalent reference data for dogs. We obtained reference RI data from 22 clinically healthy dogs <4 months of age and 33 healthy dogs between 4 months and 7 years of age. An association between the RI and plasma renin activity was investigated. The mean RI in the older dogs was 0.65 +/- 0.05 vs. 0.75 +/- 0.05 in dogs <4 months of age. The mean plasma renin activity in the older dogs was 1.18 +/- 1.03 vs. 4.23 +/- 3.09 ng/ml/h in dogs <4 months of age. There was a weak linear relationship between the RI and plasma renin activity (r2 = 0.280, P < 0.01) in dogs <4 months of age. Also in these younger dogs, RI was negatively correlated with age (r2 = 0.682, P < 0.01). The RI was higher in dogs <4 months of age than in older dogs. Therefore, the mean renal RI is slightly higher in young dogs than reported for an older population and interpretation of the RI must include an assessment of patient age.

This work explored how the perception of violin notes is influenced by the magnitude of the applied vibrato and by the level of damping of the violin resonance modes. Damping influences the "peakiness" of the frequency response, and vibrato interacts with this peakiness by producing fluctuations in spectral content as well as in frequency and amplitude. Initially, it was shown that thresholds for detecting a change in vibrato amplitude were independent of body damping, and thresholds for detecting a change in body damping were independent of vibrato amplitude. A study of perceptual similarity using triadic comparison showed that vibrato amplitude and damping were largely perceived as independent dimensions. A series of listening tests was conducted employing synthesized, recorded, or live performance to probe perceptual responses in terms of "liveliness" and preference. The results do not support the conclusion that liveliness results from the combination of the use of vibrato and a "peaky" violin response. Judgments based on listening to single notes showed inconsistent patterns for liveliness, while preferences were highest for damping that was slightly less than for a reference (real) violin. In contrast, judgments by players based on many notes showed preference for damping close to the reference value.

Outbreaks of food-associated renal failure in pets occurred in Asia and the United States of America in 2004 and 2007. They were related to the combined intoxication of cyanuric acid and melamine. Our aims were to investigate cyanuric acid and melamine contamination of pet food and to examine subchronic toxicity in rats. Levels of 10%, 20%, 50%, and 50%-100% (w/w) of contaminated pet food were fed to rats for three months. Analytical results revealed that the tainted food contained significant levels of cyanuric acid and melamine in a ratio of 1:6.8. Rats fed the diet of 50%-100% for three months exhibited elevated serum blood urea nitrogen and creatinine, as well as dose-dependent melamine/cyanuric acid crystal-induced nephrotoxicity. The melamine/cyanuric acid crystals of various sizes were mixed with necrotic cell debris and inflammatory cells, accompanied by tubular dilation and interstitial fibrosis. The immunohistochemistry index of proliferative cellular nuclear antigen and osteopontin in the kidney of the 50%-100% group were elevated, indicating regeneration of renal cells and the formation of crystals. In conclusion, the combination ratio of cyanuric acid to melamine and the acidic urine content were two factors that, upon repeated exposure, determined the severity of the nephrotoxicity.

The purpose of this article was to investigate the effects of sedatives and general anesthetics, such as tiletamine-zolazepam, medetomidine, and isoflurane on the short ERG protocol. Six healthy mongrel dogs were assessed by a convenient short ERG protocol with the owners' consent. The amplitudes of a-wave and b-wave, as well as the implicit time of ERG under different anesthesia statuses, were recorded and analyzed. The amplitudes of ERG waves were not significantly different between tiletamine-zolazepam and medetomidine groups, except in b-wave after 5 min dark adaptation (140 +/- 42 microV in tiletamine-zolazepam and 101 +/- 32 microV in medetomidine, p<0.01). The amplitude of ERG recorded in isoflurane (5 +/- 3 microV of a-wave and 12 +/- 6 microV of b-wave under light adaptation; 41 +/- 19 microV of b-wave after 1 min dark adaptation; 28 +/- 15 microV of a-wave and 58 +/- 32 microV of b-wave after 5 min dark adaptation) were significantly different from tiletamine-zolazepam (8 +/- 2 microV of a-wave and 24 +/- 9 microV of b-wave under light adaptation; 117 +/- 44 microV of b-wave after 1 min dark adaptation; 59 +/- 18 microV of a-wave and 140 +/- 42 microV of b-wave after 5 min dark adaptation), except in a-wave after 1 min dark adaptation (39 +/- 13 microV in tiletamine-zolazepam and 34 +/- 17 microV in isoflurane). Comment-General anesthesia had significantly lower amplitudes in the dark-adapted group compared with the sedation group. Therefore, tiletamine-zolazepam is a desirable choice for the short ERG protocol in dogs.

The objective of this study was to explore the feasibility of intestinal epithelial cell and human source vector used in gene therapy for hemophilia B. The intestinal epithelial sw480 cells were transfected with human source vector plasmid pHrnF9 which contained human coagulation factor IX gene. Transcription of its mRNA were measured by RT-PCR. The transfection efficiency were observed under fluorescence microscope. The expression of its protein and coagulant activities in the transfected sw480 cells were measured by ELISA and one-stage method. The results showed that the expression of hFIX mRNA could be detected after transfection. The transfection efficiency reached to the maximum at 48 hour. The hFIX protein amount was 11.3 +/- 0.23 ng/(10(6) cells.24 h) at 24 hours after transfection and reached to 29.34 +/- 1.00 ng/(10(6) cells.24 h) at 48 hours and decreased to 12.45 +/- 0.15 ng/(10(6) cells.24 hr) at 72 hours. Sw480 cells transfected with pHrnF9 were capable of producing hFIX with coagulant activity. The coagulant activity reached to (6.07 +/- 0.17)%/10(6) cells at 48 hours and decreased to 1.81 +/- 0.06%/10(6) cells at 72 hours. It is concluded that the sw480 cells transfected with pHrnF9 plasmid can express hFIX with coagulant activity, the intestinal epithelial cells may become target cells in the gene therapy for hemophilia B.

An 11-year-old neutered male domestic shorthair cat was presented with an epulis. A hemispherical mass, 8mm in maximum diameter, without a peduncle and bright reddish in colour, was observed on the gingiva of the left mandible. Radiography failed to show any infiltrating osteolysis. The epulis was surgically removed via gingival incision around the margin to the depth of connective tissue layer. Histopathological examination indicated that the epulis contained a large number of multinucleated giant cells (MGCs) intermixed with mononuclear mesenchymal cells in a loose fibrovascular stroma. Mitotic cells were found, mainly in the centre of the mass. MGCs were stained positive by the tartrase resistant acid phosphatase (TRAP) staining, indicating osteoclasts activity. Immunohistochemical staining for proliferating-cell nuclear antigen (PCNA) was observed within the majority of mononucleated cells, whereas multinucleated cells did not stain. An osteoclast-like giant cell tumour was concluded in this case. The origin of epulis is likely from the periosteal tissue. The cat recovered uneventfully and no recurrence has been noted for 3 years thereafter.

Objective: To explore the different therapeutic effects of comminuted clavicular fracture with acromioclavicular external fixtatior and DCP internal fixation., Methods: There were 768 cases of comminuted clavicular fracture involved in the study. Among them, 528 patients (321 male and 207 female, aged from 15 to 82 years) treated with acromioclavicular external fixator, in which there were 165 cases of three parts fracture and 363 cases of more than three parts fracture; 240 patients (152 males and 88 females, aged from 17 to 64 years) treated with the internal fixation, in which there were 178 cases of three parts fracture and 62 cases of more than three parts fracture. The time between injury and treatment was 1.3 days (range, 2 h to 8 days). The results were evaluated according to Neer scoring system., Results: All the cases were followed up from 8 to 24 months. For treatment of fracture more than three parts, there was significant difference between acromioclavicular external fixation group and internal fixation group in nonunion rate (chi2=44.17, P<0.05) and in Neer scores (t=5.284, P<0.05)., Conclusion: Treatment with DCP internal fixation which matching the AO principles can obtain anatomic reduction, firm fixation and early functional exercise; however, treatment with self-designed acromioclavicular external fixator is an ideal therapeutic method as it has, early union of the fracture and good functional outcome with seldom complications.

A randomized single-blind trial was conducted to evaluate the efficacy of short-term abdominal acupuncture as a novel treatment for insomnia in Chinese women. Forty-four patients between the ages of 22 and 56 were randomly assigned to an acupuncture (n = 23) and a medication group (n = 21). The acupuncture group received abdominal acupuncture once a day for the first three days and once every three days for the remaining 11 days. In addition, every subject in acupuncture group also received a placebo pill once daily. Abdominal acupuncture was administered according to a standardized protocol involving four master and four adjunctive acupoints: Zhongwan (CV 12), Xiawan (CV 10), Guanyuan (CV 4), and Qihai (CV 6); bilateral Shangqu (KI 17), Huaroumen (ST 24), Xiafengshidian, and Qipang. Subjects in the medication group were treated with sham acupuncture at the same time as the acupuncture group and received estazolam once a day. The outcome measure was the Leeds Sleep Evaluation Questionnaire (LSEQ), administered before and after the trial. Subjects who received abdominal acupuncture lowered their LSEQ scores by an average of 26.32 points (95% CI: 37.34, 15.30). After controlling for potential confounding factors, the effect of abdominal acupuncture in relieving insomnia was still statistically significant. Results indicate that short-term abdominal acupuncture is more effective than pharmacological treatment for relieving insomnia in adult women and has few adverse effects.

Objective: Antikinectin autoantibody has recently been identified as a potential biomarker in Behçet's disease (BD). Here, we established an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescent assay (IFA) for detecting this antibody. The clinical significance of antikinectin was investigated., Methods: Partial or full-length cloning for human kinectin in prokaryotic or eukaryotic system was carried out. Three fragments covered kinectin coding sequence were used to establish ELISA. Full-length kinectin overexpressed HepG2 cells were used as a substrate for IFA. Serum samples from BD (n = 46), systemic lupus erythematosus SLE, n = 16), rheumatoid arthritis (RA, n = 160, ankylosing spondylitis (AS, n = 14), primary Sjörgen syndrome (pSS, n = 12), mixed connective tissue disease (MCTD, n = 8), and healthy donors (n = 51) were examined., Results: Good measurement consistency between IFA and ELISA (p < 0.001) and previous immunoprecipitation assay (p = 0.011) was found. Antikinectin was found not only in 32.6% (IFA) to 41.3% (ELISA) BD patients but was also identified in pSS, SLE, MCTD, and RA with prevalence ranging from 12.5% to 25%. Nevertheless, the titer of antikinectin (ELISA) is statistically higher in BD compared to other autoimmune connective tissue diseases (p = 0.0286). Antikinectin was found exclusively among complete form of BD (p < 0.001), but there was no correlation with specific clinical manifestations., Conclusions: We confirmed the previous observation that antikinectin is related to BD, especially in the complete form of disease. The specificity and predictive values are moderate.

Isospora michaelbakeri is one of the Isospora species most commonly found in the wild field, which can cause severe infection and mortality in young sparrows. In this study, we selected I. michaelbakeri (Chung Hsing strain) as a pathogen to orally inoculate russet sparrows (Passer rutilans), spotted munia (Lonchura punctulata), canary (Serinus canaria), Java sparrows (Padda oryzivora), chicken (Gallus domesticus), ducks (Anas platyrhynchos) and BALB/c mice. The results indicated that I. michaelbakeri infected only russet sparrows. Infected sparrows displayed lethargy, muscular weakness and fluffy feathers, followed by rapid death. Liver and spleen enlargement was seen in the infected birds. Schizonts were identified in thin smears from the venous blood, enlarged livers and spleens. Histopathological examination revealed schizonts and merozoites from the liver and spleen of infected russet sparrows, but not from other species experimentally inoculated with I. michaelbakeri in the present study.

Formosan gem-faced civets are classified to be endemic sub-species of Paguma larvata in Taiwan. Little about their reproductive physiology has been reported. This study was designed to characterize the ovarian activity throughout the year and define ovarian cycle length and the lengths of its component phases. Serum samples were collected for enzyme immunoassay (progesterone and estradiol) from seven captive civets twice weekly for 1 year. Meanwhile, periodic changes in external genitalia (vulva swelling) and vaginal cytology were examined and recorded. Results showed estrous cycles exhibited two types: 18-day (18.5+/-1.1, n=64) and 28-day (27.6+/-1.0, n=28) as shown by progesterone and estradiol fluctuations and corresponding changes in vulva morphology and vaginal cytology. Both types showed a similar 7-day follicular phase, peaking progesterone at Day 7. The 18-day cycle type prevails in the spring and summer whereas the 28-day cycle type is significant in the autumn. In summary, female gem-faced civets are polyestrous (approximately 13 cycles/year), and non-typical seasonal breeders, with follicular phase and two distinct durations of luteal phases (diestrus) cycling throughout the year, but the frequency of ovarian cycles was remarkably gradually decreased from September to February of next year. Zoo Biol 0:1-11, 2007. (c) 2007 Wiley-Liss, Inc.

Objective: To observe the effects of Guben Huatan Tongmai Recipe (GBHTTMR), a compound Chinese herbal recipe, on expressions of macrophages and cell adhesion molecules (CAMs) of aortic endothelia in rats with syndrome of phlegm blocking blood vessel, and to explore the pathogenesis of the phlegm-pathogen., Methods: Fifty normal male Wistar rats, 7-week in age, were randomly divided into five groups: normal control group, untreated group, high-dose GBHTTMR-treated group, low-dose GBHTTMR-treated group and simvastatin-treated group, with 10 rats in each group. Syndrome of phlegm blocking blood vessel was induced in rats of the latter 4 groups by feeding the rats with high lipid diet. Levels of blood lipid were compared among the 5 groups. The expressions of macrophages and CAMs in aortic endothelia were tested by immunohistochemical staining method., Results: The level of blood lipid, and the expressions of macrophages and CAMs showed statistical differences between the normal control group and the untreated group (P<0.01), and between the untreated group and the low-, high-dose GBHTTMR-treated and simvastatin-treated groups as well (P<0.05)., Conclusions: GBHTTMR can decrease the level of serum cholesterol and triglycerides, and increase the level of high density lipoprotein. It also can inhibit the expressions of macrophages, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, P-selectin, and E-selectin.

Sperm acrosome reaction (AR) is a prerequisite step for in vivo fertilization. In the vicinity of the oocyte, zona protein(s) (ZP) and progesterone (P4), a component of follicular fluid, are proven to be responsible for physiological AR induction. In the present study, a thorough analysis of the role of the progesterone receptor (PR) in this processing including in vitro physiological studies and biochemical isolation and characterization of the receptor protein was conducted. Following capacitation for 0, 2, 4 and 6h, pooled fertile boar semen samples (n=6) with >70% sperm motility were labeled with P4-BSA-FITC (100 microg/ml) to detect the activation of PR. Parallel sperm samples were treated with P4 (10 microg/ml) for 20 min to test AR inducing efficiency at different time points. To compare the ability of ZP and P4 to induce AR, spermatozoa capacitated in a modified medium supplemented with 1mg/ml heparin for 4h, were then treated with heat solubilized ZP (150 microg/ml), P4 (10 microg/ml) or ZP+P4 for 20 min. FITC-peanut agglutinin staining was applied to observe the disrupt acrosomal morphology. A purification protocol for crude boar sperm membrane proteins was developed based on ligand-receptor affinity chromatography procedures. The PR proteins were then identified by using mAb C262 raised against intracellular PR, combined with second antibody (SDS-PAGE, Western blotting). Their N-terminal amino acid sequence was determined. The amount of PR-activated spermatozoa was enhanced with time (onset: 27+/-5%, 2h: 41+/-4%, 4h: 49+/-3% and 6h: 52+/-4%, mean+/-S.E., n=6) as evidenced by increasing percentage of spermatozoa with completed cap fluorescent staining. In parallel sperm samples, percentages of AR induced by P4 were 9+/-2, 14+/-2, 18+/-2, and 24+/-2%, respectively. In solvent control at all time points, less than 10% spermatozoa had undergone AR. Capacitation for 4h or greater time periods resulted in optimal percentage of PR-activated and acrosome-reacted spermatozoa. After sperm incubation in heparin-medium, ZP+P4 treatment induced greater amounts of AR than either P4 or ZP alone (13+/-1% compared with 8+/-1 and 10+/-1%, P<0.01). Inducing capacity of P4 was comparable to that of ZP. The molecule weights of two apparent PR molecular masses were detected to be at Mr 74 kDa and Mr 63 kDa. N-terminal amino acid sequence of 74 kDa protein was XPXNIVLIFADXLXY, which had 78% homology to arylsulfatase A and 88% homology to 72 kDa protein from boar spermatozoa. The activation of PR is associated with the capacitating process and that appears to be required for P4-induced AR. P4 and ZP appear to be equally capable of independently inducing the AR but lack synergetic or additive effects in this induction process. Both might represent alternative pathways thus resulting in alternative systems for induction of the prerequisite acrosomal exocytosis (supported by NSC 90-2313-B-005-114; 91-2313-B-005-131).

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

Objective: To introduce Menopause-Specific Quality of Life (MENQOL) into China, and to evaluate the quality of its Chinese Version., Methods: MENQOL was translated into Chinese language and the reverse translation was done by several specialists. After retest study on 30 cases, validity, reliability and responsiveness were implemented in 409 cases with menopause syndrome in the multicenter study., Results: 7 components were extracted in factor analysis, and the total cumulative contribution was 59.476%. By correlation analysis, 7 components were divided into four domains: vasomotor, psychosocial, physical and sexual items, same as in the English Version. The test-retest reliability of four domains were 0.806, 0.804, 0.941, 0.940 respectively. Cronbach alpha of four domains were 0.7258, 0.8234, 0.8475, 0.8641 respectively. There had been significant changes after treatment noticed., Conclusion: The measurement properties of MENQOL-Chinese Version including effectueness, reliability were met with satisfaction and seemed to be adaptable to Chinese menopause women.

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone-bovine serum albumin-fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 +/- 4%), but this labelling was markedly reduced (33 +/- 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 +/- 10% (onset) to 59 +/- 7% by 5 h, where it plateaued. Progesterone (P 4 ) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 ug/mL P 4 corresponding to 10 +/- 5%, 16 +/- 9%, 23 +/- 7% and 30 +/- 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P 4 -induced AR (12 +/- 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P 4 -induced AR (10 microg/mL) in capacitated ejaculated spermatozoa from 19 +/- 6% to 11 +/- 4% (h151, 1 : 10) and 12 +/- 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P 4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

In Taiwan, Setaria digitata infection causes a lumber paralysis in increasing number of cattle. Culex quinquefasciatus is one of the predominant mosquitoes, and it has been suspected that C. quinquefasciatus acts as a vector to Setaria nematodes prevalence but this was not confirmed. C. quinquefasciatus, Aedes albopictus and A. aegypti of various strains were investigated using an artificial infection system to evaluate their vector competence. After blood feeding at day 14, the number of larvae (stage III) per infected mosquito in A. aegypti (Liverpool strain), A. aegypti (Kaohsiung strain), A. aegypti (Tungan strain), C. quinquefasciatus (Taichung strain) and A. albopictus (Taichung strain) was 1.3 +/- 0.1, 1.3 +/- 0.1, 1.4 +/- 0.1, 1.0 +/- 0.0 and 0 +/- 0.0 (mean +/- S.E.M), respectively. The vector efficiency index of A. aegypti (Liverpool) was the highest among mosquitoes whereas A. albopictus showed a complete refractoriness to the infection. In conclusion, C. quinquefasciatus demonstrates its potential competence for serving as a transmission vector of S. digitata. This mosquito might therefore be responsible, at least in part, for the prevalence of cattle lumbar paralysis in Taiwan. This is the first report of C. quinquefasciatu demonstrating its vector competence for S. digitata.

This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen (n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5+/-0.4 ml, 77+/-6% and 1471.3+/-940.0 x 10(6) ml(-1), respectively. Post-thaw motility in respective extender was A: 66+/-16%; B: 71+/-2%; C: 73+/-6%; D: 9+/-4% and E: 26+/-12% (mean+/-S.D.). In extender C (74+/-14%) more viable spermatozoa were preserved than in the others (A: 64+/-10%; B: 48+/-11%; D: 41+/-16%; E: 47+/-6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used (P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3+/-0.5 ml, 82+/-4% and 379.1+/-252.2 x 10(6) ml(-1), respectively. Post-thaw motility was in respective extenders A: 69+/-2%; B: 74+/-6%; C: 73+/-2%; D: 13+/-6% and E: 31+/-20%. Extenders B and C were superior (P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70+/-7%), B (76+/-7%) and C (79+/-2%) was higher than that D (25+/-19%) and E (29+/-17%) (P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86+/-4%) and C (83+/-4%) than in extenders A (54+/-13%), D (39+/-22%) and E (46+/-22%) (P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation (P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk-Tris-Tes-glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk-Tris-citric acid-glycerol based extender B containing Equex and extender C are superior in semen cryopreservation to others for Formosan Sambar deer.

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors., (Copyright 2000 Wiley-Liss, Inc.)

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Subfertility in stallions is attributed to the inability of spermatozoa to undergo the acrosome reaction in response to progesterone. In the present study, it was assessed whether there is a correlation between stallion fertility, defined on the basis of first cycle foaling rate and first cycle 'non-return rate', and the proportion of spermatozoa with exposed progesterone receptors on their plasma membranes. Semen from Dutch Warmblood (n=10) and Friesian (n=4) stallions was analysed. Progesterone 3-(o-carboxymethyl) oxime-BSA coupled with fluorescein isothiocyanate was used as a progesterone receptor probe and ethidium homodimer was used as a supravital stain. A high correlation was observed between the proportion of spermatozoa with exposed progesterone receptors and stallion fertility (r > 0.70; P < 0.01). This result indicates that exposure of progesterone receptors is a potential parameter for predicting stallion fertility.

The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellular progesterone receptor was conducted to validate the presence of the progesterone receptor. Immunogold labeling techniques enabled ultrastructural localization of P-BSA-FITC or C-262 with transmission electron microscopy. The dynamic changes in labeling patterns were monitored for sperm cells, using fluorescence microscopy and flow cytometry during a 5-h capacitation period. An increasing number of viable cells showed affinity for P-BSA-FITC or C-262 at the acrosomal plasma membrane region of the sperm head, while a decreasing number of viable cells were not labeled. In contrast, almost all deteriorated cells were labeled in the cytosol of the postequatorial region of the sperm head. Incubation with P-BSA-FITC resulted in the induction of AR but to a lesser extent than that for sperm incubated with free progesterone. Therefore, coupling of progesterone to its receptor on the sperm plasma membrane appears to be an important step in the induction of the AR.

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa. In some species, the sperm undergo the acrosome reaction before binding to the zona pellucida, and in other species only acrosome-intact sperm can initiate binding to the zona. In the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of hemizonae was similar. Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 microm2 of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.

The sensitivity of the plasma membrane to calcium ionophore (A23187) challenge was studied in dog sperm using fluorescein lectin staining for the assessment of acrosomal status and viability. Second fraction ejaculates from 5 dogs were washed, resuspended in Ca(2+)-free (EDTA-treated), 50, 100, 500, 1000 and 2000 microM/l Ca(2+)-containing Sp-TALP medium and induced with 50, 250, 500, 1000, 2500 and 5000 nM/l calcium ionophore. Samples were collected from each aliquot after 30 and 60 min of induction to assess the percentage of acrosome reacted sperm cells (AR rate), viability and motility by fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA) and ethidium-homodimer combined staining. On each slide, 200 sperm cells were assessed under epifluorescence microscope (x 1250) in a blind manner. The response to ionophore challenge (AR rate, viability, motility) varied with Ca2+ and ionophore concentration in the suspension. A significantly higher AR rate was detected in samples containing 100, 500, 1000 and 2000 microM/L Ca2+ (> 40%) than in that containing 50 microM/L. Acrosome reaction could not be successfully induced in the EDTA-treated sample and in any of the aliquots in which 50, 250 and 500 nM/L ionophore concentrations were used for induction. Motility decreased drastically in all of the treated samples and stopped in that sample where as significant AR rate could be detected. Viability remained high (> 75%) during the incubation and did not differ significantly in the treated and the control groups.

Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.