Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen–Thawed Stallion Spermatozoa

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E™). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen–thawed semen samples.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

In vitro Fertilizing Capacity of Frozen-thawed Bull Spermatozoa Selected by Single-layer (Glycidoxypropyltrimethoxysilane) Silane-coated Silica Colloidal Centrifugation

Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll™ (C2). Sperm recovery surpassed 50% for both C1–C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p < 0.05). In T, the mean (±SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 ± 23.3%, 24.5 ± 14.3% and 94.6 ± 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.     

Seminal Plasma Characteristics and Expression of ATP‐binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability

The composition of seminal plasma and the localization of the ATP‐binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen–thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm‐rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC‐PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post‐thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow‐up studies revealed whether a relation exists between these findings.     

Localization of CD9 Molecule on Bull Spermatozoa: Its Involvement in the Sperm–Egg Interaction

Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA‐50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen‐thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti‐CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.     

公牛家系泌乳曲线的研究

本文根据北京黑白花奶牛２２个公牛家系的女儿泌期资料，利用Ｗｏｏｄ模型估计了各公牛家系的泌乳曲线参数，并且按照家系内女儿的不同胎次，所在牧场和产犊年份分组作了更加深入的研究。所考虑的曲线参数为规模因子ａ。产奶量上升率ｂ和下降率ｃ；次级参数包括３０５天实际产奶量，３０５天估计产奶量，高峰月份，高峰月产奶量和泌乳持久力。结果表明，与低产公牛家系相比，高产公牛家系的泌乳曲线通常具有较高的初始产奶量，参数ａ     

Normal Swallowing in the Dog: A Cineradiographic Study

Normal dog swallowing dynamics were analyzed and correlated with specific structures. There were oropharyngeal, esophageal, and gastroesophageal phases. The oropharyngeal phase was characterized by food prehension and bolus formation (oral stage), reflex pharyngeal peristalsis (pharyngeal stage), and reflex cricopharyngeal sphincter relaxation (cricopharyngeal stage). The esophageal phase was characterized by primary peristalsis initiated by the oropharyngeal phase and secondary peristalsis which occurred in response to mechanical stimuli from a bolus in the esophagus. There was a post-peristalsis refractory period during which new peristalsis would not occur. Repeated rapid swallowing would, therefore, result in accumulation of ingesta in the esophagus. The gastroesophageal phase occurred when a bolus passed through the gastroesophageal junction, an area which serves as a lower esophageal sphincter. Gastroesophageal reflux was observed as a normal, infrequent event followed by rapid esophageal clearance. Low doses of tranquilizers had no significant influence on swallowing function. The differences observed between sternal and lateral recumbent postures related only to the rate of food consumption which, in turn, influenced esophageal motor response and inhibited gastroesophageal closure. These differences did not adversely affect the autonomous phases of swallowing.     

大夏学堂在信息素养教育中的运用——以华东师大《文献调研的策略与方法》课程为例

大夏学堂网络教学平台综合了多媒体和网络技术的功能。利用大夏学堂构建《文献调研的策略与方法》课程进行课堂教学,并以学习小组的方式进行协同学习,有利于个性化自主学习环境定制,有助于学生协同学习活动的开展,能够提高教学活动效率。     

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.     

Conserving the Greater Sage-Grouse: A Social-Ecological Systems Case Study from the California-Nevada Region

The Endangered Species Act (ESA) continues to serve as one of the most powerful and contested federal legislative mandates for conservation. In the midst of heated debates, researchers, policy makers, and conservation practitioners champion the importance of cooperative conservation and social-ecological systems approaches, which forge partnerships at multiple levels and scales to address complex ecosystem challenges. However, few real-world examples exist to demonstrate how multifaceted collaborations among stakeholders who share a common goal of conserving at-risk species may be nested within a systems framework to achieve social and ecological goals. Here, we present a case study of Greater Sage-grouse (Centrocercus urophasianus) conservation efforts in the “Bi-State” region of California and Nevada, United States. Using key-informant interviews, we explored dimensions and drivers of this landscape-scale conservation effort. Three themes emerged from the interviews, including 1) ESA action was transformed into opportunity for system-wide conservation; 2) a diverse, locally based partnership anchored collaboration and engagement across multiple levels and scales; and 3) best-available science combined with local knowledge led to “certainty of effectiveness and implementation”—the criteria used by the US Fish and Wildlife Service to evaluate conservation efforts when making listing decisions. Ultimately, collaborative conservation through multistakeholder engagement at various levels and scales led to proactive planning and implementation of conservation measures and precluded the need for an ESA listing of the Bi-State population of Greater Sage-grouse. This article presents a potent example of how a systems approach integrating policy, management, and learning can be used to successfully overcome the conflict-laden and “wicked” challenges that surround at-risk species conservation.     

Analysing Motility Parameters on Fresh Bull Semen Could Help to Predict Resistance to Freezing: A Preliminary Study

Batches of straws often need to be thrown away after freezing because of a too few number of motile or progressive sperm cells after thawing. Our objective was to evaluate the possibility to predict before freezing the quality of the semen after freezing/thawing. Computer‐Aided Sperm Analysis was performed on motility parameters both before and after freezing of 20 ejaculates from different bulls. Significant variation between bulls was observed both before and after freezing for all the analysed traits (anova 2; p < 0.001): proportion of motile (%mot) and progressive (%prog) sperm, velocity on the curved line (VCL), velocity on the straight line (VSL), velocity on the average path (VAP), linearity (VSL/VCL), beat cross frequency and average orientation change of the head. Freezing significantly altered the motility parameters and correlations were found between samples analysed before and after freezing (Pearson coefficient: R = 0.43–0.72; p < 0.05). The mean VAP, VSL and the %prog obtained before freezing were highly correlated to the %mot and %prog observed after freezing (R = 0.75–0.82; p < 0.001). Applying thresholds on mean VAP and VSL values allowed us to predict respectively 6 and 7 of nine batches that would be rejected after freezing due to a too low %prog (<15%). Combining different traits did not add to the precision. In conclusion, analysis of velocity traits on fresh sperm seems more efficient than analysis of %mot or %prog to discard batches that will be of poor quality after freezing.     

DNA Status on Thawed Semen from Fighting Bull: A Comparison Between the SCD and the SCSA Tests

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37°C with and without oxidative stress (1 m m FE²⁺). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE²⁺ treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI∼10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.     

Lethal Acrodermatitis in Bull Terriers: A Problem of Defective Zinc Metabolism

Abstract— Three bull terrier pups from a litter of six developed exfoliative, crusty areas of alopecia, particularly affecting the feet and head, by the time they were seven weeks of age. Biopsies from two of the affected pups revealed parakeratotic hyperkeratosis consistent with zinc deficiency, but the dogs did not respond to oral zinc supplements. The breed, history and dermatopathology were considered consistent with lethal acrodermatitis of bull terriers, a condition that appears to be familial and autosomally recessive. Whereas similar acrodermatitic conditions in humans (acrodermatitis enteropathica) and cattle (lethal trait A46 of Black Pied Danish cattle) respond to oral zinc supplements, this condition in bull terriers may be due to an error in zinc metabolism at the cellular level. Attempts to treat one of the pups parenterally with zinc resulted in death after the fourth injection. Résumé— 3 choits d'une portée de 6 Bull Terriers ont développéà l'âge de 7 semaines une dermite podale et faciale exfoliative, crouteuse et alopécique. Les biopsies cutanées pratiquées sur 2 chiots ont révélé une hyperkératose parakératosique compatible avec une carence en zinc, mais les animaux n'ont pas répondu à une supplémentation en zinc. La race, l'anamnèse et l'aspect histologique, sont compatibles avec une acrondermatite léthale du Bull Terrier, maladie familiale, transmise sur un mode autosomal récessif. Des acrodermatites similaires chez l'homme (acrodermatitis enteropatica) et les bovins (anomalie léthale A46 de la Pie Noire Danoise) répondant à une supplémentation en zinc, cette affection chez le Bull Terrier pourrait êtro à une erreur dans le métabolisme du zinc au niveau cellulaire. Un essai de traitement d'un des chiots par vole parcutérale avec du zinc a abouti à sa mort après la quatrième injection. Zusammenfassung— Drei von sechs Bullterrierwelpen aus einem Wurf entwickelten im Alter von 7 Wochen exfoliative, krustige und haarlose Bezirke, besonders an Pfoten und Kopf. Biopsien von zwei der betroffenen Welpen zeigten eine Parakeratose, die bei Zinkmangel auftritt. Die Tiere sprachen jedoch nicht auf orale Zinksubstitution an. Rasse, Anamnese und Befunde der Hautuntersuchung stimmen mit der letalen Akrodermatitis der Bullterrier überein, einem familiären, autosomal rezessiv erblichen Leiden. Während ähnliche akrodermatitische Erkrankungen beim Menschen (Akrodermatitis enteropathica) und beim Rind (Letalfaktor A46 beim schwarzgefleckten Dänischen Rind) auf orale Zinkgaben ansprechen, ist diese Krankheit beim Bullterrier wahrscheinlich auf eine Störung im Zinkmetabolismus auf zellulärer Ebene zurückzuführen. Der Versuch, einen der Welpen parenteral mit Zink zu behandeln, führte zum Tod des Tieres nach der vierten Injektion. Resumen Tres cachorros de Bull Terrier de una camada de seis desarrollaron areas de alopecia exfoliativa, particularmente afectando a los pies y la cabeza, para el momento en que tenian siete semanas de edad. Biopsias de dos de los cachorros afectados revelaron hiperqueratosis paraqueratótica con deficiencia de zinc, pero los perros no respondían a los suplementos de zinc oral. La raza, historia y dermatopatología se consideraron consistentes en acrodermatitis letal de Bull Terriers, una condición que parece ser familial y autosómica recesiva. Mientras que condiciones de acrodermatitis similares en humanos (acrodermatitis enteropética) y ganado (rasgo letal A46 del ganado Black Pied Danish) respondían a los suplementos de zinc oral, esta condición en los Bull Terriers puede ser debida a un error en el metabolismo del zinc a nivel celular. Intentos de trater uno de los cachorros, por vía parenteral con zinc resultaron con la muerte después de la cuarta inyección.     

A Surgical Approach to the Ramus of the Mandible in Cattle and Horses Case Reports of a Bull and a Horse

A surgical approach to the ramus of the mandible was developed. Subperiosteal elevation of the masseter muscle from the ramus allowed reduction and plate fixation of ramus fractures in a bull and a horse. Results of follow-up examinations, 2 years after surgery, showed excellent functional and cosmetic results.     

Nuisance and Odor Issues in Broiler Production: A Case Study

Recent expansion of the broiler industry in Texas resulted in an opportunity to experience and mitigate a number of nuisance and odor issues related to broiler production. For the sake of this discussion, the participants in these issues are divided into 3 groups: the poultry industry; the nonpoultry citizenry; and government, regulatory, and educational agencies. Establishment of a broiler complex in an area with little exposure to commercial poultry production resulted in a concerned nonpoultry citizenry. This was often exacerbated by misinformation, external influences, and poor communications, resulting in a less-than-optimum level of trust between the nonpoultry citizenry and the poultry industry. Early in the process, the poultry industry attempted to provide information and open communication with the nonpoultry citizenry; however, establishment of bias within the nonpoultry citizenry eroded trust and cooperation to the point that effective communication was difficult. Throughout this period, government, regulatory, and educational agencies were involved in investigations of complaints, facilitation of communication, and development of policy. Over a 2-yr period these activities resulted in a special legislative appropriation to investigate odor and nuisance issues related to broiler production. This investigation and other agency activities resulted in the development of regulatory policy that required all broiler producers to develop comprehensive nutrient management plans. Although these plans are primarily concerned with water quality and environmental issues, the nonpoultry citizenry was willing to accept this step as a means to assure that poultry facilities would be operated in compliance with best management practices. As a result, the poultry industry now had specific policy and guidelines for dealing with environmental issues, and the nonpoultry citizenry had assurance that the broiler operations would be held to a standard of operation. At this time, the level of communication and trust between all parties is greatly improved.     

草地反射太阳光偏振特性测量个例分析

本文在中国科学院植的研究所岩石草原定位研究站对羊草和苔草样方进行太阳反射光偏振特性的测量。