Literature concerning the time frames for detection of various seminal components commonly tested for in forensic laboratories in sexual assault cases is limited in quantity and in scope. Determining a more accurate time since intercourse (TSI) interval based on an extensive review of forensic case work would provide investigators with a tool for estimating the time elapsed between the occurrence of a sexual assault and the collection of a Sexual Assault Evidence Collection Kit (SAECK) which could be vital information in certain cases. This study demonstrates that the presence of intact spermatozoa is a significant finding on microscope slides prepared from vaginal, anorectal, and oral swabs and that the percentage of intact sperm cells decreases over time. This study also proved that sperm tails are lost during the preparation of microscope slides from SAECK swabs by directly comparing medical personnel-prepared smear slides and analyst-prepared extract slides from 95 Boston Police Department (BPD) Crime Laboratory Unit cases. Additionally, this study presents maximum TSI values for the persistence of sperm heads, intact spermatozoa, and prostate specific antigen (P30) through a retrospective examination of 355 cases processed by the BPD Crime Laboratory Unit over 5 years. The maximum persistence values for P30 in the vaginal, anorectal, and oral cavities were 19 hours, 17 hours, and 20 hours, respectively. In the vaginal cavity, maximum persistence values for intact spermatozoa were 43 hours for smear slides and 41.5 hours for extract slides. The maximum persistence of sperm heads was equivalent for vaginal smear and extract slides at 105 hours. In the anorectal cavity, maximum persistence values for intact spermatozoa were 43 hours for smear slides and 13 hours for extract slides. The maximum persistence of sperm heads was equivalent for anorectal smear and extract slides at 43 hours. In the oral cavity, maximum persistence values for intact spermatozoa were 3.75 hours for smear slides and 5 hours for extract slides. The maximum persistence of sperm heads were equivalent for oral smear and extract slides at 24 hours.

Determina el efecto de la criopreservación sobre la integridad acrosomal de los espermatozoides viables de alpaca recuperados del epidídimo, determinado mediante citometría de flujo. Se procesaron muestras de 46 testículos de alpaca obtenidos de Camal Municipal de Ninacaca, Pasco, que presentaron una motilidad ≥ 30% y concentración espermática ≥ 50x106 espermatozoides/mL, en promedio 46.6% y 61x106 espermatozoides/mL, respectivamente del total de las muestras obtenidas. Los espermatozoides se recuperaron de la cola del epidídimo con 1mL de dilutor a base de leche descremada, yema de huevo, fructuosa y DMA; separándose luego en 2 alícuotas de 500 μL. La primera alícuota se utilizó para la evaluación de la viabilidad e integridad acrosomal inicial y la segunda alícuota se congeló en pajillas mediante un sistema automático de criopreservación, para luego almacenarse en nitrógeno líquido hasta el día de su evaluación. Todas las muestras, frescas y descongeladas fueron lavadas 2 veces por centrifugación con solución PBS para retirar el dilutor. Para la evaluación de viabilidad e integridad acrosomal, 100μL de cada muestra fue incubada con 2.5μL de FITC-PSA (100 μg/mL) y 0.5μL de Ioduro de Propidio (PI, 2.4 nM) por 10 minutos a 38°C, obteniendo finalmente una concentración de 2.5 μg/mL y 12 μM, respectivamente. Inmediatamente después las muestras se evaluaron por citometría de flujo con analizador de imágenes, adquiriéndose diez mil eventos compatibles con espermatozoides por muestra. FITC-PSA y PI fueron excitados con el láser de 488 nm, mientras que la emisión de fluorescencia fue detectada utilizando los canales Ch02 (505-560 nm) para FITC-PSA y Ch05 (642-740nm) para PI. Se seleccionó la población de espermatozoides viables (PI negativos), que en promedio fue el 75% del semen fresco y un 42% del semen descongelado, para de ellos determinar el porcentaje de integridad acrosomal (FITC-PSA negativos) y de reacción acrosomal (FITC-PSA positivos). Se realizó un análisis de T-student pareado para determinar como la criopreservación afecta la integridad acrosomal en los espermatozoides viables. Se encontró que la integridad acrosomal de los espermatozoides viables inicial (98.25 ± 5.40%) fue similar (p>0.05) a la integridad acrosomal de los espermatozoides viables post descongelamiento (98.75 ± 2.17%). Se concluye que si bien la criopreservación disminuye la cantidad de espermatozoides viables, la estructura acrosomal no se altera en los espermatozoides de alpaca que sobreviven a este proceso. / Trabajo de suficiencia profesional

El objetivo del estudio fue evaluar el efecto de cuatro combinaciones de dos agentes crioprotectores permeantes más dos no permeantes sobre la calidad del semen ovino post-descongelamiento. Para esto, primero (Experimento 1) se seleccionó, entre tres dilutores (A, B y C), el más adecuado para ser usado en la segunda parte (Experimento 2), en la cual se evaluaron las siguientes combinaciones de agentes crioprotectores permeantes y no permeantes: 1)Glicerol - Trehalosa, 2)Glicerol - Sacarosa, 3)Etilenglicol - Trehalosa y 4)Etilenglicol - Sacarosa sobre la calidad del semen ovino post-descongelamiento. Para tal efecto, se realizaron 6 repeticiones para el Experimento 1 y 8 repeticiones para el Experimento 2. Cada repetición constó de 4 eyaculados cada una. En el experimento 1 se encontró que el Dilutor A mantenía mejor la motilidad progresiva y viabilidad e integridad acrosomal post-descongelamiento (69% y 63%) en comparación con el Dilutor B (37% y 35%) y el Dilutor C (23% y 23%). Por lo cual, el Dilutor A fue empleado en el experimento 2. En el experimento 2 se encontró que la motilidad progresiva, la viabilidad e integridad acrosomal, la termoresistencia y la integridad de membrana plasmática post-descongelamiento fue mejor en los grupos con glicerol-sacarosa (64%; 56%; 40% y 63%) y glicerol-trehalosa (64%; 58%; 38% y 60%) en comparación con los grupos con etilenglicol-sacarosa (48%; 42%; 25% y 37%) y etilenglicol-trehalosa (44%; 38%; 27% y 40%). Lo cual demuestra que el glicerol es un mejor crioprotector permeante en comparacion con el etilenglicol. Sin embargo, no existe diferencia en el uso de sacarosa o trehalosa. Se concluye que un dilutor con las características del dilutor A, utilizando glicerol más trehalosa o sacarosa constituye una buena alternativa para la criopreservación de semen ovino. / --- The objective of the study was to evaluate the effect of four combinations of two permeant and two non permeant cryoprotectant agents on the quality of post thaw ram semen. In Experiment 1, three extender were evaluated (A, B, and C) in order to have th ebetter one for the next step. In Experiment 2, different combinations of cryoprotectant agents were evaluated as follow: 1) Glycerol–Trehalose, 2) Glycerol–Sucrose, 3) Ethyleneglycol-Trehalose, and 4) Ethyleneglycol–Sucrose. In this way, 6 assays for Experiment 1 and 8 assays for the Experiment 2 were carry out. In each assay, 4 ejaculated were mixed to work as an original sample. Percentages of motility, viability and acrosomal integrity in extender A were higher (69% and 63%) than extender B (37% and 35%) and extender C (23% and 23%). Therefore, extender A was used for experiment 2. In the experiment 2, motility, viability and acrosomal integrity, thermoresistance, and plasmatic membrane integrity were higher in groups Glycerol-sucrose (64%; 56%; 40%, and 63%) and Glycerol-trehalose (64%; 58%; 38%, and 60%) in comparison with groups Ethileneglycol-sucrose (48%; 42%; 25% and 37%) and Ethileneglycol-trehalose (44%; 38%; 27% and 40%). However, there is not significative differences between sucrose or trehalose. In conclusion, an extender with characteristics of extender A, it means, using glycerol plus trehalose or sucrose constitute a good alternative for cryopreservation of ram semen. / Tesis

Semen is one of the most common biological fluids encountered by a forensic serologist on varying substrate types. Seminal fluid contains many enzymes, proteins, and cellular material such as acid phosphatase (AP), prostate-specific antigen (PSA), semenogelin (Sg), and spermatozoa; detection of these components can aid in the forensic identification of body fluids. Forensic laboratories usually follow a prescribed testing workflow (from visual examination including an alternate light source (ALS) and AP testing to PSA or Sg testing followed by a microscopic examination for sperm cells) to ensure laboratory resources are being used in a proper manner with minimal waste of both time and resources. However, this approach can be problematic when degradation of semen stains results in the inability to detect the presence of certain seminal components. When a stain yields negative results for an AP reaction, no further analyses for semen may be performed and analysis comes to an end. In common practice, evidentiary items containing biological fluids may not be immediately recovered following an incident and/or may not be stored properly, causing contamination or exposure of these biological fluids to harsh environments, potentially degrading the sample. This study investigates how exposure to different environmental conditions and packaging types affects the degradation of the four most common semen components targeted in forensic testing: AP, PSA, Sg, and spermatozoa. Semen stains were prepared and exposed to ten different storage and/or environmental conditions to compare their effects on the detectability of seminal components (fluorescence, AP, PSA, Sg, and spermatozoa as well as deoxyribonucleic acid (DNA) testing) for a period of approximately four months. Samples from each condition were tested on select days throughout the study. Minimal changes in detection of the seminal components were observed under the five conditions in which the stains remained dry: packaged in paper stored at room temperature, packaged in paper stored at high temperatures, exposed to sunlight, exposed to ultraviolet light, and stored in high humidity. Two of the conditions involved exposure to outdoor environments. The stains openly exposed to the elements or buried in soil exhibited the most notable degradation of all components when compared to other conditions. Negative results were obtained for nearly all seminal components (AP, PSA, or Sg) on Day 8 for stains openly exposed outdoors and Day 32 for buried samples. The remaining three conditions exposed the stains to damp or wet conditions and gave variable results throughout the study. DNA quantification was performed for select samples from each condition to assess DNA degradation. Most samples did not exhibit DNA degradation on quantification results up to Day 112; however, two samples exposed to outdoor environments exhibited DNA degradation as early as Day 8 (earliest day quantified). More notably, two samples from Day 112 demonstrated the presence of non-degraded DNA in sufficient quantity for profiling, while the presumptive semen analyses (AP, PSA, and Sg) for the same samples exhibited negative results when using an AP reaction cut-off time of 2 minutes. These results suggest that an allotted time of 2 minutes for AP detection may not be sufficient in some samples, and that valuable DNA evidence may go undiscovered, especially when other presumptive tests show negative results. Overall, the results revealed variation in the sequence and rate of degradation for seminal components in semen stains exposed to different environmental conditions. It was not possible to predict which of the remaining components of semen would be detectable based on the outcome of any one of the tests. Therefore, it is recommended that comprehensive testing of possible semen stains is performed, even after negative presumptive results are obtained, when the case scenario suggests exposure to damp/wet or otherwise less than ideal conditions.

Alternatives to antibiotic growth promoters have been widely exploited due to concerns about antimicrobial resistance. These feed additives improve growth, in part, by modulating intestinal microbiota. However, their impact on male reproductive performance is not well elucidated. Therefore, the objective of this study was to evaluate the impacts of a yeast fermentation product (YP) and Bacillus subtilis on rooster semen quality and microbiota. Dietary supplementation of YP linearly increased the concentration of yeast and bacteria in semen, whereas it linearly decreased sperm motility, suggesting that bacteria attached to yeast were excreted from the gut, contaminated semen at the cloaca and then decreased sperm movement. However, direct in vitro exposure of semen or dietary supplementation with B. subtilis did not affect semen quality or seminal concentration of this bacterium, likely because Bacillus naturally occur in semen. In conclusion, unlike B. subtilis, dietary YP can alter semen quality by altering semen microbiota.

First, a preliminary study was conducted looking for the optimum sperm concentration to be used for analysis with the Computer-Assisted Sperm Analysis (CASA). Results showed that 75x106 sperm cells/mL is the optimum one. Then, the actions of relaxin on sperm motility were evaluated by determining the effect of relaxin on full motility characteristics of spermatozoa during storage, using CASA; then identifying the relaxin receptors on spermatozoa, and finally establishing actions of relaxin in intraspermatic cAMP content. Motile spermatozoa were selected through percoll gradient and incubated for 1 hour with 4 relaxin concentrations at 37°C, during four days. Relaxin affected sperm motility (P<0.05). This action appears associated with the presence of relaxin receptors RXFP1 and RXFP2 that were found in spermatozoa. However, the cAMP levels were not affected by relaxin (P<0.05). This study indicates a beneficial action of relaxin on sperm motility; however, its mechanism of action requires further research.

No description available.

This study occurred in the Rural Academic Unit-Tiahuanaco installations, of the Bolivian Catholic University-La Paz, Bolviia, with the objective of evaluating macro- and microscopic characteristics of sperm ejaculation from bulbourethrectomized llamas. Six q'ara-variety male llamas of 3, 4, and 5 years of age were used over 8 weeks during which they were fed with natural and cultivated pastures. Ejaculate was collected with an artificial vagina with stimulation (libido) of male llamas by female llamas, for macro- and microscopic evaluation (volume, pH, color, appearance, motility, concentration, and sperm vitality). The results obtained were: average volume of 0.55[+or-]0.36 ml, with a CV of 20.2%; average pH of 7.53[+or-]0.42 with a CV of 4.9%; the ejaculate's color varied between crystal white, opaque white, and milky white at proportions of 50%, 25%, and 25%, respectively; average motility was 25.9[+or-]21.8% with a CV of 27.6%; average sperm concentration was 28.7x106[+or-]20.11x106 sperm/ml with a CV of 13%; average live sperm count was 31.8[+or-]24.4% with a CV of 25.3%; and the ejaculate an appearance of nonviscous fluid. The 4-year-old animals had excellent sperm ejaculations (macro- and microscopic characteristics) without differences between collection weeks.

No description available.

Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science