Penicillin and amikacin mixture has bactericidal activity equivalent to gentamicin, tylosin, lincomycin and spectinomycin mixture in equine frozen semen.

Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, a bioequivalence study was performed to assess the bactericidal activity of GTLS and PA in equine frozen semen. Nine mature, healthy stallions were used in the study. Split ejaculates were processed using media without antibiotics (Control) or with different antibiotics. For the GTLS group, centrifugation medium and freezing extender were prepared with gentamicin 250 µg/ml, tylosin 50 µg/ml, lincomycin 150 µg/ml and spectinomycin 300 µg/ml. For the PA group, the centrifugation medium was prepared with potassium penicillin G (PPG) 1200 units/ml and the freezing extender was prepared with PPG 1200 units/ml and amikacin 500 µg/ml. Semen processed in extenders without antibiotics had higher (p < .005) bacterial loads throughout all cryopreservation processing steps than semen samples processed using antibiotics. There were no differences in semen bacterial load after centrifugation, 15 and 30 min after final extension, and after thawing between GTLS and PA groups, but PA had faster (p < .05) kill-time kinetics than GTLS. Only minor differences in sperm kinetic parameters were observed among groups. In conclusion, this study demonstrated bioequivalence between GTLS and PA in mitigating end-point bacterial loads. Prudent concentrations of the antibiotic mixtures evaluated in this study can be considered both effective and sperm-safe for equine frozen semen.

Principles and Clinical Uses of Real-Time Ultrasonography in Female Swine Reproduction.

Within the past 30 years, through ongoing technology and portability developments, real-time (b-mode) ultrasonography (RTU) has increasingly become a valuable diagnostic tool in assessing the female reproductive tract in swine. Initially applied in swine production to visually determine pregnancy status, RTU use has expanded to include assessment of the peri-pubertal and mature non-pregnant females as well. Transabdominal and transrectal modalities to visualizing the reproductive tract in swine have been reported with the transabdominal approach more common due to the fact of its ease of accessibility, animal/personnel safety, and reduced time to perform. Adjustable frequency transducers are preferred as they allow optimization of image quality at various depths. If a single transducer frequency must be selected, a 5 MHz probe provides the best versatility for visualizing the reproductive tract in swine. Other basic requirements for ultrasound equipment which will be used on commercial swine farms include being light weight and easy to handle, readily cleanable and disinfectable, long battery-life, and good durability. When using RTU for pregnancy determination, diagnosis is based upon a combination of the animal's breeding records, the presence of embryonic fluid, and, depending upon gestational stage, fetal structures. If RTU is used as a diagnostic tool in assessing reproductive problems in an individual or a group of animals, sonographic evaluation of both the uterus and ovaries is performed. Tissues are delineated and assessed based upon their echogenicity, echotexture, and size. Uses of RTU in clinical practice may include assessment of delayed puberty, prolonged wean-to-estrus interval, absence of post-weaning estrus, herd disruptions in conception and farrowing rates, vulval discharge, peripartum and puerperal disorders. This review aims to provide an overview on principles and clinical uses of RTU with respect to application to address female reproductive performance issues in commercial swine operations.

Andrology laboratory review: Evaluation of sperm concentration.

This article is the result of the work of the andrology task-force of the Association of Applied Animal Andrology, American College of Theriogenologists, European College of Animal Reproduction, Society for Theriogenology, and National Association of Animal Breeders. It is intended to serve as a comprehensive reference on methods to evaluate sperm concentration and to contribute to the adoption of best practices in veterinary andrology laboratories. The information covered in the article includes sample preparation and the use of manual counts, spectrophotometers, computer-assisted semen analysis, NucleoCounter, and flow cytometry. Emphasis is given to the principles of the methods and equipment, performing the evaluation, and common mistakes and/or pitfalls. In addition, the precision and accuracy of the different methods are also discussed.

Testing an egg yolk supplemented diet on boars to aid in sperm adaptation at 5°C.

In many species, extended semen can be stored at low temperatures to slow bacterial growth. However, boar semen performs poorly at temperatures below 15 °C and this poses unique challenges, as it is not easy to maintain a constant 15-19 °C during shipment. Some extenders have been formulated with egg yolk for storage at 5 °C but the addition of egg yolk is not applicable in the majority of commercial operations. The purpose of this study was to evaluate if boar dietary supplementation with powdered egg yolk imparts any protective effects on sperm quality when stored at 15 °C and 5 °C for up to 11 days in a conventional extender. Ten boars were fed a commercial diet with the addition of 0.11 Kg of powdered egg yolk for 10 weeks. Ejaculates collected on weeks 4, 6, 8, and 10 were processed for storage at both 15 °C and 5 °C and compared with ejaculates from boars fed a standard diet. Throughout an 11-day storage period, sperm quality was assessed including several motility and morphologic parameters and select plasma membrane properties (fluidity, integrity, and triacylglycerol content). Linear regression models were used to describe effects of treatment, storage day, week and temperature on all sperm parameters. Overall, there were minimal beneficial effects of egg yolk treatment on sperm quality parameters. Sperm from egg yolk supplemented boars did have a slower decline in viability and plasma membrane fluidity than that observed in the control sperm when stored at 5 °C (p < 0.001). Additionally, there was an increase in total morphologic abnormalities in sperm from egg yolk fed boars compared to controls at week 10 (p < .001). In conclusion, the results of this study do not support a significant benefit to sperm quality or resistance to cold storage when feeding a 10-week dietary supplementation of 0.11 Kg powdered egg yolk to crossbred boars.

Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm.

Capacitation is a biochemical pathway sperm must undergo to be able to fertilize an oocyte, whereas cryoinjury is cryopreservation-induced biophysical damage which renders sperm immediately capable of fertilization. Similarities between capacitation and cryoinjury have not been fully elucidated. The present study attempted to characterize both processes, including the role of seminal plasma (SP). Merocyanine-540 staining detected an increase (P < 0.01) in plasma membrane disorder from 60.5% in in vitro capacitated sperm to 91.4% in cryopreserved sperm, with no effect of SP. After cryopreservation, 42.8% of sperm displayed phosphatidylserine on the outer leaflet compared to 13.6% of in vitro capacitated sperm (P < 0.01), as assessed by annexin-V staining (SP decreased phosphatidylserine inversion in both populations). Lipid raft-associated glycolipid GM(1) movement increased throughout the entire sperm membrane in cryopreserved sperm, although SP did not affect lipid raft movement in these sperm. Cryopreserved and in vitro capacitated sperm had a similar intensity of tyrosine phosphorylation (although SP reduced this intensity). In in vitro capacitated sperm, 67.5% underwent an ionophore induced acrosome reaction with 91.3% reacting in cryopreserved sperm. In both cases, SP reduced (P < 0.01) the percentage of acrosome-reacted sperm to 1.0 and 7.8%, respectively. Cryopreservation appeared to damage sperm, resulting in marked increases in membrane disorder, cholesterol efflux, and percent of capacitated sperm. In both capacitated and cryoinjured sperm, the addition of SP appeared to attenuate some of these events.

Prevalence of malignancy when solitary versus multiple lesions are detected during abdominal ultrasonographic examination of dogs with spontaneous hemoperitoneum: 31 cases (2003-2008).

OBJECTIVE: To compare the histopathologic diagnosis in dogs with spontaneous hemoperitoneum when abdominal ultrasonographic examination detects a solitary versus multiple lesions. DESIGN: Retrospective cross-sectional study. SETTING: Private veterinary hospital. ANIMALS: Client-owned dogs presented with spontaneous hemoperitoneum between March 1, 2003 and June 1, 2008. INTERVENTIONS: Dogs were divided into 2 groups based on presence of a solitary or multiple abdominal ultrasonographic lesions. Prevalences were compared between groups for malignancy and specifically hemangiosarcoma. MEASUREMENTS AND MAIN RESULTS: Ten of 31 (32%) dogs had a solitary abdominal ultrasonographic lesion and 21 of 31 (68%) had more than 1 lesion. The bleeding tissue was characterized as malignant in 8 of 10 (80%) dogs with solitary lesions and 17 of 21 (81%) dogs with multiple lesions; there was no significant difference (P=1.0) between groups. In this study no association (P=0.26) was found between the number of abdominal ultrasonographic lesions observed and subsequent diagnosis of hemangiosarcoma. CONCLUSIONS: Solitary abdominal ultrasonographic lesions in dogs with spontaneous hemoperitoneum do not necessarily indicate a lower prevalence of malignancy.

Comparative antiviral and proviral factors in semen and vaccines for preventing viral dissemination from the male reproductive tract and semen.

Many animal and human viruses are disseminated via semen, but there is little information on how to measure and stimulate protective antiviral immunity in the male reproductive tract and semen. This information is important since successful vaccination through the stimulation of protective immune responses could be a mechanism to prevent viral contamination of semen and subsequent wide spread viral dissemination. Even control of the infection by shortening the duration of viral shedding and lowering the viral load in semen would lessen the chances of viral dissemination through this route. This review will highlight the current knowledge of immunity in the male reproductive tract and summarize 'antiviral' as well as 'proviral' factors in semen such as cytokines, cells, antibodies, antimicrobial peptides, enzymes, hormones and growth factors. These factors must provide a fine balance between 'immunosuppression' in semen needed to protect sperm viability and 'immunocompetency' to prevent pathogen contamination. The review will also suggest continuing challenges to researchers for preventing viral dissemination via semen and propose a large animal model for continued research in this important area.

Current concepts of molecular events during bovine and porcine spermatozoa capacitation.

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.

Bacteriospermia in extended porcine semen.

Bacteriospermia is a frequent finding in freshly extended porcine semen and can result in detrimental effects on semen quality and longevity if left uncontrolled. The primary source of bacterial contamination is the boar. Other sources that have been identified include environment, personnel, and the water used for extender preparation. A 1-year retrospective study was performed on submissions of extended porcine semen for routine quality control bacteriological screening at the University of Pennsylvania. Out of 250 sample submissions, 78 (31.2%) tested positive for bacterial contamination. The most popular contaminants included Enterococcus spp. (20.5%), Stenotrophomonas maltophilia (15.4%), Alcaligenes xylosoxidans (10.3%), Serratia marcescens (10.3%), Acinetobacter lwoffi (7.7%), Escherichia coli (6.4%), Pseudomonas spp. (6.4%), and others (23.0%). Prudent individual hygiene, good overall sanitation, and regular monitoring can contribute greatly in controlling bacterial load. Strategies that incorporate temperature-dependent bacterial growth and hyperthermic augmentation of antimicrobial activity are valuable for effective control of susceptible bacterial loads. Aminoglycosides remain the most popular antimicrobial class used in porcine semen extenders, with beta-lactam and lincosamide use increasing. With the advent of more novel antimicrobial selection and semen extender compositions in swine, prudent application and understanding of in vitro pharmacodynamics are becoming paramount to industry success in the use of this breeding modality.

Particle distribution in low-volume capillary-loaded chambers.

Accurate determination of sperm concentration in fluid suspension is a critical component in a semen analysis. Inaccurate estimations can lead to misinterpretation of the spermiogram and, in the case of livestock production, can lead to faulty insemination doses, which can adversely affect stud power, fertility, fecundity, and cost effectiveness of breeding programs. Capillary-loaded slides, like the hemacytometer, have been the standard for calibration of other concentration estimation modalities such as photometry, Coulter counter, flow cytometry, and computer-automated semen analysis (CASA). Single-use capillary-loaded slides, much smaller than the hemacytometer, are frequently used by many of the current CASA systems. As the use of CASA increases, more field reports are suggesting differences between CASA results and hemacytometry. In this article, we establish that these differences are, in large part, due to the Segre-Silberberg effect, which occurs during Poiseuille flow in high-gradient fluid flow in thin capillary-loaded slides. We develop the theory of this phenomenon and derive the scaling and significance of the effect. Finally, we graphically provide a means for predicting the necessary compensation factor when using capillary-loaded slides to determine sperm concentration.

Capillary-loaded particle fluid dynamics: effect on estimation of sperm concentration.

Capillary loaded chambers are frequently used for semen analysis. Poiseuille flow of specimen into these chambers causes migration of suspended particles or cells in a direction transverse to the flow, which results in their preferential accumulation in the Segre-Silberberg (SS) planes. This SS effect depends on the transverse velocity gradient in the laminar flow. For semen analysis in thin capillary-loaded slides, the SS effect can lead to erroneous estimation of sample sperm-cell concentration. To better understand chamber flow dynamics and SS effect significance, we assessed flow uniformity, inflow cell velocity, and results of concentration measurements under different flow conditions for latex bead and porcine and human sperm suspensions. Overall, a concentration peak was present at the meniscus, which continued through chamber loading. High-velocity SS preferred planes, which channeled particles toward the meniscus, were located at the fractional positions of beta = .27 and beta = .73, where beta is the distance from wall to plane normalized to the chamber depth. In computer-automated semen analysis, a standard 20-microm x 18-mm x 6-mm chamber is commonly used, and these studies supported our previously published fluid-flow theory for this type of chamber. Conversely, the SS effect does not appear to have time to develop in the 100-microm-depth hemacytometer, which is deeper than the standard slide, has lower transverse velocity gradient, and consequently does not exhibit concentration variation due to the SS effect. These findings provide further support that hemacytometry, when performed properly, remains the gold standard. Applicability of our findings to routine semen analyses was then tested in 2 studies performed with independent boar studs. These studies compared diluted boar semen concentrations estimated by standard hemacytometry and in capillary-loaded 20-microm slides, using a computer-automated semen-analysis system designed to compensate for the SS effect. Good numerical agreement for sperm concentration with a high degree of correlation (r(2) = .936) was found between the 2 techniques. These findings reaffirm the need to critically assess new technologies for accuracy, repeatability, and precision.

Immunofluorescence reveals ubiquitination of retained distal cytoplasmic droplets on ejaculated porcine spermatozoa.

The purpose of the present study was twofold: 1) to determine if antibodies raised against ubiquitin would recognize antigens associated with the porcine cytoplasmic droplet (CD), and 2) to determine if the same antibody would identify ubiquitinated substrates on the surface of morphologically abnormal boar spermatozoa. Permeabilization with the detergent Triton X-100 (0.05%) showed virtually all CDs to be ubiquitin positive. Distal droplets (DDs) retained in situ on boar spermatozoa were readily labeled following Triton permeabilization, whereas DDs present on nonpermeabilized cells were not. Negative control preparations lacked the ubiquitin staining on the DD. The use of microtubes for fixation and incubation provided clearer images as well as better sperm cell distribution and density than an initial slide-mounted technique. Immunoblotting indicated that larger amounts of ubiquitinated proteins were present in extracts from sperm cells from an ejaculate with an abnormally high percentage of retained DDs (52% DDs) compared to a morphologically normal sample (6% DDs). The primary antibody recognized both mono-ubiquitin of bovine origin (8.5 kd) and human ubiquitin conjugate (35 kd), as demonstrated by Western blot. Preabsorption of the anti-ubiquitin antibody with purified bovine ubiquitin was successful in preventing diaminobenzidine staining of sperm extract from the high DD ejaculate. The presence of antigens recognized by anti-ubiquitin antibodies in the boar sperm CD, coupled with the possibility that superfluous ubiquitin species are detrimental to embryonic development by targeting critical paternally contributed zygotic organelles, raises concerns that retained DDs may be more detrimental to fertility than previously suspected.

Concentrations of endogenous prostaglandin F2alpha in boar semen and effect of a 72-h incubation period on exogenous prostaglandin F2alpha concentration in extended boar semen.

PGF2alpha in semen has been shown to induce uterine contractions, thereby, facilitating sperm transport during fertilization. Previously, we demonstrated that extended boar semen used in artificial insemination does not increase myometrial contractility, but PGF2alpha supplementation did. In this study, we determined the concentrations of endogenous PGF2alpha in pre-sperm and sperm-rich fractions of the boar ejaculate and examined whether changes in the concentration of exogenous PGF2alpha occurred when added to extended boar semen after 72-h incubation at a 17 degrees C storage temperature. Concentrations of endogenous PGF2alpha (n = 10 boars) in pre-sperm and sperm-rich fractions were 69.6 +/- 7.6 and 58.9 +/- 4.4 pg/ml, respectively. No differences were observed in the concentrations of exogenous PGF2alpha in the extended boar semen at 0 h (59.3 +/- 3.3 microg/ml) and after a 72-h incubation period (52.0 +/- 2.1 microg/ml). These results suggest that the concentration of endogenous PGF2alpha in boar semen used for artificial insemination is < 100 pg/ml. The concentration of exogenous PGF2alpha in the extended boar semen did not differ after 72 h, which indicates that it is not metabolized during this period of time.