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There is an increased demand for artificial insemination and shipping canine semen in clinical practice. However, we need to process the semen samples using centrifugation and dilution with extenders to help preserve the breeding dose and semen quality. Our objective was to determine a clinically relevant range of centrifugation and concentration parameters for processing canine semen. In the first experiment, we hypothesized that higher g force and longer treatment improves sperm recovery rates yet causes greater decline in semen parameters over a 48-hour cooling period. Our study design used the raw semen evaluations which served as each dog's own control. Sperm RR (%) was calculated post-centrifugation, and sperm viability (%, Nucleocounter® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (NM%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 (T2) and 48 hours (T3) after cooling. Sperm losses were minimal and similar for all treatment groups (median >98%, P≥0.062). Spermatozoa viability was not different between centrifugation groups at any time point (P≥0.38) but declined significantly during cooling (T1 vs. T2/T3, P≤0.001). Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 (P≤0.02). In conclusion, our study showed that centrifugation within a range of 400g-900g for 5-10 minutes is appropriate for processing canine semen. In the second phase, we compared different sperm concentrations for cooled canine semen storage and hypothesized that lower concentrations would result in better semen quality. Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 x106 sperm/ml (C200), then serially diluted to 100, 50, and 25 x106 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24h, then centrifuged and re-extended. Parameters were assessed in raw semen (T0), post-extension (T1), after 24h of cooling (T2), and after processing at 24h (T3). Cooling resulted in significant declines in STM and NM for all groups, and in decreased PMI for CON and C25-50. After cooling (at T2), PMI was significantly lower for C25 compared to all groups and higher for CON compared to C25-100 (p≤0.038). For the motility parameters and NM, C25 performed worse than all or most of the other groups. Comparing CON at T3 with C25-200 at T2, PMI, STM and NM for CON were significantly lower than C25-200, C200, and C100-200, respectively. In conclusion, our results show that cooling canine semen for 24h at 200 x106 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on highest PMI. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24h of cooled storage.

This study analysed the reservoir of HIV-1 in semen and the effects of seminal plasma on the functional and phenotypic characteristics of dendritic cells (DCs), both in relation to immunoregulatory capabilities and susceptibility to infection with HIV. The reservoir of HIV-1 in semen was defined in order to assess the feasibility of 'sperm washing' as a means of reducing the risk of transmission in HTV-1-discordant couples. The fact that neither viral RNA or DNA could be detected in spermatozoa and the lack of expression of CD4 both at the protein and mRNA levels suggests that spermatozoa are not susceptible to HTV-1 infection. Viral RNA was detected in seminal plasma and both RNA and DNA were detected in non-sperm cells. As a result of this study, sperm washing is a service that is now available at the Chelsea & Westminster Hospital to HTV-l-discordant couples. This service has resulted in several births and no seroconversions in any woman who has undergone the procedure. The effects of seminal plasma on the phenotype and function of monocyte-derived DCs were investigated. Seminal plasma had suppressive effects on the allostimulatory capacity of DCs and such effects appeared to be due to down- regulation of co-stimulatory molecule expression on these cells. The suppressive effect of seminal plasma was abrogated by the removal of lipids. However, prostaglandins on their own did not have suppressive effects on DC function, suggesting that other seminal components are required to induce theobserved induction of suppression. The suppressive effect of seminal plasma on the allostimulatory function of DCs was overcome with TNF-a, as did the presence of HIV. The expression of HIV co-receptors was assessed on DCs. TNF-a induced maturation of DCs as demonstrated by reduced expression of CCR5 and increased CXCR4. Seminal plasma also enhanced expression of CXCR4 and infection with X4 strains of HIV, but down-regulated CCR5 expression and infection with R5 strains of HIV. Therefore, the regulation of co-receptor expression by DCs was associated with their susceptibility to infection with a HIV strain displaying the corresponding co-receptor usage. In summary, semen is an important vehicle for transmission of HIV. Seminal plasma was demonstrated to have profound effects on DC phenotype, maturation and function. Such alterations would influence the outcome, in terms of HIV transmission, of the interaction between the DC and HIV-1 at the mucosal surface.

Sexual assault evidence composes a large portion of the evidence analyzed by forensic serologists. Key to the processing of sexual assault evidence is the screening of the evidence items for the presence of semen. Due to the intimate nature of a sexual assault, it is very possible that semen is mixed with other body fluids when it is deposited on an item of evidence. One of the body fluids that semen can easily come into contact with during a sexual assault is saliva. Saliva functions as the first step in the human body’s digestive system. Due to the digestive system’s purpose of breaking down nutrients, it stands to reason that saliva could play a role in breaking down seminal components. To detect semen, specific components of semen are tested for in forensic laboratories. These components are often acid phosphatase, prostate specific antigen, semenogelin, and spermatozoa. This experiment combined semen from one donor with saliva of seven other donors in a two part survey. In part one, semen was mixed with the saliva of Donors A, B, C, and D at three different ratios of 1:1, 1:2, and 1:10 semen-saliva. Twenty-microliter stains were pipetted onto one inch by one inch squares on twelve cotton swatches to test for acid phosphatase, prostate specific antigen, semenogelin, and spermatozoa. One set of six swatches was allowed to dry and the other set was kept damp. The stains were tested at six timepoints: day zero, day one, week one, week two, week three, and week four. Part two involved incubating the semen with saliva from Donors X, Y, and Z at body temperature for up to twenty-four hours. The same three ratios used in part one were repeated with the saliva from Donors X, Y, and Z in part two. A twenty microliter stain was pipetted onto a cotton swatch at each of the five timepoints from the start of the incubation period: zero minutes, one hour, five hours, eight hours, and twenty-four hours. Each stain was tested for acid phosphatase, prostate specific antigen, and semenogelin. The results of part one showed that semen samples that are mixed with saliva but allowed to dry are effectively unaffected by the presence of saliva. On the dry swatches, the stains tested positive for every component of semen at every timepoint for every donor except for Donor D’s 1:10 semen-saliva mixture stain, which tested negative for spermatozoa at week 1, but positive for spermatozoa in the subsequent timepoints. The results of the damp swatches suggests that damp environmental factors can prevent the detection of seminal components. By week two, the detection of spermatozoa had completely dropped out in the mixture stains and in the neat semen control stains. Detection of prostate specific antigen ceased in the control by week 3 and had also stopped in all 1:1 semen-saliva mixture stains by week 4. The detection of prostate specific antigen had stopped at week 3 for all donors in the 1:10 semen-saliva mixture stains. Semenogelin was still detectable in the control sample for the duration of the experiment, and it was detected for all donors at week 4 in the 1:1 and 1:2 semen-saliva mixture stains. Detection of semenogelin ceased in Donors B and D in the 1:10 semen-saliva mixture stains by week two. The results of part two suggested that the detection of acid phosphatase could be affected when semen and saliva have been incubating together at body temperature. Acid phosphatase was detected at all the timepoints in the neat semen control, but after eight hours, it was no longer detectable in all the mixture ratios of Donors X and Z. Acid phosphatase was no longer detectable in the 1:1 semen-saliva mixture stain of donor Y at eight hours and in the 1:2 semen-saliva mixture stain of Donor Y at twenty-four hours. Acid phosphatase did remain detectable in the 1:10 semen-saliva mixture stain of Donor Y through the twenty-four hour experimental period. Prostate specific antigen and semenogelin remained detectable in all the donors at all three ratios for the duration of the experiment.

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.

Nutritional management and semen collection method are thought to influence semen quality parameters evaluated during a breeding soundness examination. Therefore, the objective of this experiment was to evaluate the effects of two feeding strategies on growth performance and semen quality of developing bulls. A secondary objective was to quantify the effects of two semen collection methods on semen quality. Angus bulls (n = 48; 332 ± 47 d of age) were stratified into one of sixteen drylot pens before random assignment to one of two iso-caloric diets: a total mixed ration (TMR; n = 8 pens) or a component-based ration (COMP; n = 8 pens). Bulls were assigned to either electroejaculation (EE; n = 24) or trans-rectal massage (RM; n = 24) and each collection method occurred twice throughout an 84-d feeding period. Rumen pH was continuously monitored via rumen boluses and dry matter intake (DMI) was measured daily. Backfat thickness and hoof conformation were measured on d 0 and 84, with body weight measured and semen collected at 21-d intervals. Semen was assessed for progressive motility, total sperm per ejaculate, and sperm cell morphology. Bulls fed the COMP ration had a greater DMI (P < 0.01) and ADG (P < 0.01), but similar feed efficiency (P = 0.19) when compared to TMR-fed bulls. Bulls fed COMP had a lower mean rumen pH (P < 0.01). Additionally, COMP-fed bulls had greater backfat thickness (P < 0.01) but similar hoof conformation scores (P ≥ 0.55) when compared to TMR-fed bulls on d 84. Bulls fed the COMP had greater semen volume (P < 0.05) but feeding management did not affect progressive motility (P = 0.42), total sperm per ejaculate (P = 0.86), or percent morphologically-normal sperm (P = 0.59). However, bulls collected via RM had greater total sperm per ejaculate (P < 0.05), and semen volume (P < 0.01), but lower percent morphologically-normal sperm (P < 0.05) and progressive motility (P < 0.01). Neither diet nor semen collection method affected BSE passage rate (P ≥ 0.34), suggesting minimal influence on BSE outcomes in developing bulls.

Studies have highlighted a growing national problem regarding the number of untested Sexual Assault Kits (SAKs). A 2011 National Institute of Justice report revealed Los Angeles alone had 10,000 untested SAKs. This backlog has fueled the need for specific and efficient testing of SAK evidence. In traditional workflows, serology tests are used to indicate the presence of a targeted bodily fluid and prioritize samples for genetic analysis. However, given the lack of sensitivity and specificity of modern serological assays, current SAK workflows often skip serological identification altogether for a “direct to DNA” approach. While these Y-Screen workflows achieve rapid screening of samples for the presence of a detectible male contributor, they do not provide any serological information. As a result, samples lack what can be critical investigative context. Improved serological capabilities with enhanced sensitivity and specificity would provide greater confidence in results for the confirmatory identification of seminal fluid. At a minimum, forensic biologists should understand the limitations associated with traditional serological approaches to seminal fluid identification when processing SAK samples. Current serological techniques based on antigen-antibody binding have exhibited both sensitivity and specificity limitations. False positive results for semen can be obtained by non-target biological fluids such as breast milk, urine, and vaginal fluid, or by non-specific binding events. This study evaluates a promising emerging technique that combines high specificity protein biomarker detection with targeted mass spectrometry. This research targeted human-specific peptide markers for seminal fluid proteins and peptide standards to perform quantification of seminal fluid peptide targets using an Agilent 6495 mass spectrometer coupled to a 1290 series liquid chromatograph. This approach has shown to be both more specific and sensitive in identifying a bodily fluid compared to current immunological based approaches. Thus, this proteomic workflow was used to evaluate authentic false positive rates of current immunochromatographic techniques for seminal fluid identification. Self-collected vaginal swabs collected from participants not engaging in barrier-free vaginal intercourse with male partners were tested using various immunochromatographic assays designed to detect both semenogelin (Sg) (RSID™-Semen) and prostate specific antigen (PSA) (ABAcard® p30 Test and SERATEC® PSA Semiquant). Similarly, three seminal fluid biomarkers (semenogelin 1, semenogelin 2, and prostate specific antigen) were used for seminal fluid identification via mass spectrometry. Any samples producing positive results on any immunochromatographic assay were evaluated to determine whether the target protein was actually present at levels above the reported sensitivity limits of the lateral flow tests. Additionally, Sperm HY-LITER™ Express was used to microscopically confirm the absence of spermatozoa in all samples producing positive immunochromatographic results. In addition to using the quantitative proteomic assay to estimate the rate of authentic false positive results associated with lateral flow assays, this research sought to establish the correlation (or lack thereof) between absolute quantitation of seminal fluid markers and the ability to successfully generate DNA profiles. Self-collected post-coital swabs from donors engaging in barrier free vaginal intercourse with male partners over varied periods of time between 1-8 days after intercourse were collected. All samples were analyzed using the quantitative seminal fluid protein mass spectrometry assay, once again targeting SgI, SgII, and PSA. Both autosomal STR profiles (GlobalFiler™) and Y-STR profiles (Yfiler™ Plus) were subsequently generated. With regard to immunochromatographic assay false positive rates, a total of 17 false positives for semen were observed (n=150), 14 of which were consistent with PSA and 3 with Sg, for a corresponding total false positive rate of 9.3% and 2%, respectively (11.3% overall). These samples were all confirmed to be sperm negative with mass spectrometry and microscopic analysis. This data supports the use of current immunochromatographic assays for the presumptive detection of seminal fluid while also providing further support for the improved specificity of alternative serological approaches using mass spectrometry identification of biological targets. With regard to the relationship between quantitative levels of target seminal fluid peptides and the ability to generate STR profiles from vaginal swabs collected at various post coital intervals, a total of 61 post-coital samples were tested. Of these, 48 samples had a seminal fluid target greater than the limit of quantitation for the mass spectrometry assay and 26 produced an STR (n=9) and/or Y-STR (n=10) profile. A correlation between peptide quantitation and ability to generate a genetic profile was unable to be determined from this initial sample set. Overall, however, it has been demonstrated that the use of proteomic mass spectrometry for the identification of seminal fluid targets (with its enhanced sensitivity and specificity) would enable forensic practitioners to make better use of serological information during the analysis of challenging sexual assault samples.

Recent research efforts in the field of forensic biology include development of a confirmatory one-step testing methodology for biological fluids. Such techniques that have been shown to be promising include the analysis of deoxyribonucleic acid (DNA) or messenger ribonucleic acid (mRNA) contained within the specialized cells of specific body fluids. In current forensic practice, the possible biological origin of an evidentiary item or stain is often evaluated using protein-based presumptive testing. Ribonucleic acid (RNA) is an effective marker for the identification of body fluids because it codes for those specific proteins within the particular cells that make up each fluid. LGC has developed the ParaDNA® Body Fluid ID test that detects specific RNA markers allowing discrimination of six different body fluids. This research focuses on PRM2 and SEMG1, indicative of spermatozoa and seminal fluid, respectively. The ParaDNA® Body Fluid ID test was investigated to evaluate its performance with forensic semen samples and to determine if it is advantageous over current conventional semen screening methodology. Factors such as sensitivity, PRM2 and SEMG1 stability in aged stains at room temperature and in semen subjected to multiple freeze-thaw cycles, and performance on post-coital swabs were evaluated. Both spermatozoa and seminal fluid were detected in one of six 1:10 diluted stains tested and zero of six 1:50 diluted stains. Conventional semen screening methodology detected both components in six trials of 1:10 and 1:50 stains. PRM2 and SEMG1 were detected in stains prepared with neat liquid semen subjected up to 10 freeze-thaw cycles, in room temperature stains stored for up to 101 days, as well as in three different post-coital swabs. Of the 82 samples run on the instrument, 22 were false negatives, with a majority of them occurring during the sensitivity study with diluted stains, indicating the ParaDNA® Body Fluid ID Test is not yet suitable to replace current conventional screening methods. Upon further validation it could serve as a valuable tool for forensic laboratories.

Typically, semen testing involves presumptive and confirmatory tests to determine the region in which a semen stain has been deposited prior to initiating DNA analysis. However, previous research showed that the soluble components of seminal fluid, but not spermatozoa, migrated from their original location on cotton cloth upon exposure to porcine decomposition fluids and rainfall/dew6. This indicates that preliminary testing and detection techniques may result in areas being sampled that will not yield a successful DNA profile. The present study assesses how various amounts of water or blood affect migration patterns of seminal fluid components using traditional serological screening methods as well as DNA analysis. The effects of exposing a semen stain to water over the course of several days are also investigated. The final component of the study evaluates whether the presence of acid phosphatase (AP) Spot reagent had any detrimental effects on subsequent antigen P30 (P30) testing, Kernechtrot Picroindigocarmine (KPIC) sperm staining or DNA analysis. Neat semen was deposited onto swatches from cotton sheets and allowed to dry before being sprayed with 2 mL, 5 mL, or 10 mL of water or blood. The swatches were allowed to dry while lying flat, at 45°, or at 90°. Three of the swatches were sprayed directly with AP Spot reagent to determine any potential interference with subsequent P30 and DNA testing. After the water or blood was dry, the swatches were viewed with an alternate light source (ALS) at 450 nm using orange barrier filter goggles. Three-millimeter fabric punches were collected from each swatch in at least thirteen locations (one from the center of the stain and four at 1 cm, 4 cm, and 7 cm from the perimeter of the stain in multiple directions), and were extracted for two hours prior to testing for the presence of P30. Additional fabric punches were collected from each P30 positive location to be used for DNA analysis. AP testing showed positive results beyond the original semen stain with an average distance of 1-3 cm from the perimeter of the original region of deposition (ORD) for all swatches except those moistened with blood. AP mapping was performed on the swatches moistened with blood and negative results were obtained. Positive P30 results were obtained for all swatches with an average distance of 1-3 cm from the ORD. The angle at which the swatch was positioned influenced the direction(s) that the soluble components migrated; however the amount of water (or blood) the swatch was exposed to had a much greater effect on the distance of migration. Microscopic examination of slides made from the extracts of each fabric punch revealed minimal spermatozoa migration for all swatches; the majority of the samples outside of the ORD showed no spermatozoa, although a few showed a single sperm cell. These findings demonstrate that the soluble components of semen stains that often aid in detection migrated when exposed to moisture, while sperm cells containing genetic material largely remained in their original location. The DNA analysis results confirmed the lack of spermatozoa migration. Full DNA profiles were obtained from within the ORD of the flat and 90° swatches. The samples from outside of the ORD produced either partial profiles (maximum dropout rate of 97%) or no profile. If case circumstances suggest that evidence has been exposed to water, multiple regions should be tested in order to maximize the possibility of identifying semen and obtaining a DNA profile. AP Spot reagent was not found to have detrimental effects on P30 testing, sperm staining or DNA analysis. Therefore, direct application of AP Spot reagent could be used for larger pieces of evidence where the location of a stain is unknown. This would eliminate the careful documentation needed for chemical mapping and the reliance on the transfer of acid phosphatase from one substrate to another.

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.

The objective was to estimate the effect of traits recorded in females and in boars and their semen on farrowing rate (FR), total number (TB) and number of stillborn pigs (SB) at birth. Results of 20,569 inseminations in 4,468 sows on 4 farms with semen from 856 boars in 2 AI centers were analyzed. Records on sows included farm, dline parity and brdeeding interval (Brdint). Records on boars included number of days rest between collections, and 26 characteristics (e.g., volume, sperm concentration, motility, abnormal heads and tails plus 16 traits that described velocity and path of sperm cell movement). At first time, we were trying to use whole boar semen traits for our analysis; however the attempt was not competitive enough to reveal which semen characteristics had been far more deeply involved in FR, TB and SB. Thus, we used STEPWISE, MAXR and R-square were used for choosing statistically best semen characteristics. Data were analyzed with SAS PROC MIXED in models accounting for fixed effects of farm dam line of sow (Dline) and parity, random effects of sow and boar, and regressions of sow reproductive traits on sow, boar, and semen traits. Models were first fitted with only linear regressions; if important (P < 0.10), 2nd models including quadratic effects were fitted. Parity and the interval from 1st insemination (1st estrous during breeding period in gilts, and 1st post-weaning estrus in sows) to the insemination that resulted in a litter affected (P < 0.01) FR, and SB (Pst insemination to insemination of conception increased from 0 to 65 days. Sow reproductive traits were not affected (P>0.10) by number of days between collections (all boars had at least 3 d rest) or sperm concentration. Ten traits (Tmot, Vol, Proximal, Distal, Compos, Head, Tail, VAP, DSL and AOC) describing semen traits affected sow reproduction (P < 0.10), but differences across the range of variation were relatively small. Advisor: Rodger K. Johnson

Two groups of bulls were utilized to evaluate if single nucleotide polymorphisms (SNP) in the Bos taurus OPN gene (GenBank accession # AY878328.1) promoter region were related to bull semen quality variables as evaluated by computer assisted semen analysis (CASA). Group 1 consisted of 19 Angus and Balancer bulls, ranging in age from 5 to 8 years, which were collected weekly during a 2-month period (July 15, 2010 through September 19, 2010). Group 2 consisted of 16 Brahman influenced (1/8 to 3/16 Brahman) bulls, mean age = 1.1 ± 0.1 year, that were collected monthly for a 7 month period (February 27, 2007 to August 16, 2007). Each collection was evaluated for motile, progressive, rapid, medium, slow, static, path velocity (VAP), progressive velocity (VSL), track speed (VCL), lateral amplitude (ALH), beat frequency (BCF), straightness (STR), linearity (LIN), elongation and area variables using CASA after semen collection. Each bull was genotyped for reported SNP in the promoter region of the OPN gene through PCR amplification of two 700 base pair fragments and sequencing of the resulting PCR product. Eight SNP sites were identified in Group 1 at base pairs 3379, 3490, 3492, 4967, 5075, 5205, 5209, and 5263 of the OPN gene and additional SNP at base pairs 3541, 3800 and 5262 in Group 2. Individual SNP sites were evaluated as the main effect on CASA measured variables in a SAS MIXED model for repeated measures. The SNP analysis found differences (P ≤ 0.05) within groups for motile, rapid, progressive, VAP, VCL, ALH, LIN and elongation variables. Haplotypes also were constructed and compared for differences in sperm variables. In Group 1, certain haplotypes appear to be more favorable for percent motile, progressive and rapid sperm, as well as VAP, VSL, VCL, elongation and total abnormalities than bulls identified with haplotype 1 (reported normal reference sequence). In Group 2, various differences were identified among haplotypes, but were only more favorable than haplotype 1 for BCF and live percentage. These results suggest that polymorphisms within the promoter region of the bovine OPN gene might be useful in certain populations for the selection of bulls with improved sperm quality.

The objectives of this study were to assess growth and reproductive performance of boars fed a diet supplemented with organic selenium (Se). Crossbred boars received one of three treatments: I. basal diet with no supplemental Se, II. basal diet supplemented with 0.3 ppm organic Se (Sel-Plex), and, III. basal diet supplemented with 0.3 ppm sodium selenite. Nursery (n = 13 pens/treatment) boar performance was not affected (P > 0.1) by diet and only grow-finish (n = 11 pens/treatment) G:F was greater (P < 0.06) for Sel-Plex (0.378) compared with selenite (0.368) or control (0.363) boars. At 15-mo of age semen was collected from boars (n = 10/treatment) over 5-d. Semen quality declined over time, but the negative impact day had on sperm motility was less pronounced with Sel-Plex boars. Effects of treatment x day were detected for progressively motile (P = 0.02) and rapidly moving (P = 0.03) spermatozoa, sperm path velocity (VAP; P = 0.05), and average velocity (VSL; P = 0.05). At 17-mo of age, semen was collected from boars (n = 10/treatment), extended and stored over 10-d. Although semen quality decreased over time, sperm from Sel-Plex boars resisted the negative effects of day on sperm motility and pH. Effects of treatment x day were detected for percent motile spermatozoa (P < 0.01), static spermatozoa (P < 0.01), VAP (P = 0.06), amplitude of head displacement (ALH; P = 0.02), straightness (P = 0.01), and pH (P < 0.01). At 23-mo of age, semen was collected (day 0) from boars (n = 6/treatment), extended, stored and evaluated at d 1 and 8 using in vitro fertilization. Dietary Se treatment failed to affect (P < 0.05) in vitro fertilizing rates of boars. In summary, dietary supplementation with Sel-Plex enhanced G:F in grow/finish boars. Dietary Sel-Plex supplementation may decrease the effects that stressors, such as intensive semen collection or semen storage, have on boar sperm characteristics such as sperm motility. The mechanisms for these responses remain to be elucidated.

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.

The ability to characterize and enhance market traits in livestock has facilitated a greater interest in determining the genetic tool kit available for manipulation. In swine, using new approaches in genomics, such as microarray analysis and biological pathway analysis, we can show genes up and down regulated in a variety of processes and conditions. To this end, we identify the genes, pathways, and disease biomarkers affected by divergent selection of testosterone in boars. Testicular samples were taken from boars at 0, 30, 120, 150, and 180 days of age for lines of high (HT) and low testosterone (LT). Evidence that many of the differences in gene expression were at the pubertal period of 150 days led to a subsequent microarray study of the 150 day HT and LT animals. Microarray studies were followed by validation with real-time RT-PCR of 11 genes and extensive GeneGo pathway analysis (Metacore) of differentially expressed genes. While increased testosterone has long been associated with increased growth rates, we now have supporting genomic evidence of the genes and pathways up-regulated and down-regulated in these lines. To this end, this study has identified several disease biomarkers that may require further investigation and biological pathways associated with growth and metabolism that allow the recommendation of selective breeding for high testosterone to increase lean growth traits. The genetic blueprint contained in the spermatozoan transcriptome can also illuminate key issues in swine reproduction. By developing a procedure for effective RNA extraction of boar spermatozoa we are one step closer to elucidating the porcine sperm transcriptome and the genes implicated in growth and fertility. A viable protocol was developed to handle the complexities of large scale extraction of RNA from porcine semen utilizing an RNA carrier and Dnase treatment. This protocol was validated with PCR amplification of Sus scrofa prm1 in order to provide evidence of a successful RNA extraction from sperm.

Swaying the bovine sex ratio is beneficial because dairy heifers are significantly more valuable than bull calves, whereas in the beef industry, bull calves are more desirable than females. Currently, there is no inexpensive, reliable, effective method for commercially separating X- and Y- bearing sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlusTM, which claims to sway the sex ratio in favor of female calves. Three trials included the insemination of hyperstimulated cows with Control or HeiferPlusTM treated semen, non-surgical embryo collection on Day 7, and a novel combined PCR/ Southern blot assay to determine embryo sex. Overall, 271 embryos were collected and a sex was assigned to 265 of them. Chi square analysis showed that the Control group produced a significantly higher proportion (p< 0.0005) of female embryos than the HeiferPlusTM (HP) group (65.0% and 43.0%, respectively). There was no difference in the proportion of transferable versus degenerate embryos and ANOVA showed no difference in the number of ovulations, embryos, and unfertilized oocytes collected from Control versus HP groups. Of the two bulls used in the hyperstimulation studies, one produced an overall higher proportion of females (p< 0.05), suggesting a bull effect. Another trial involved the insemination of cows synchronized via OvSynch¨ with fetal sexing via ultrasonography between Days 55- 58. Of the 152 cows inseminated, 51.3% (78/ 152) were pregnant at Day 35. Of the cows inseminated with HP treated semen, 54.5% became pregnant and 48.0% of the Controls were pregnant. HP treated cows resulted in 54.8% male (23/ 42) and 45.2% female (19/ 42) fetuses. Control cows had 52.8% male (19/ 36) and 47.2% female (17/ 36) fetuses at 58 days gestation. Chi square analysis showed no significant difference in the sex ratio proportions. Semen from six bulls was used in this trial, and pregnancy rates among bulls were not different. Computer automated semen analysis (CASA) showed that there was no significant difference in motility or progressive forward motility of Control versus HP treated semen in any of the bulls. Results of these studies indicated that HeiferPlusTM semen sexing kit did not sway the sex ratio in favor of females in either hyperstimulated or single-ovulating cows. In the hyperstimulated cows, the Control group actually produced a significantly higher proportion of female embryos than the HP group. No differences in embryo production or embryo grade were observed. Further research is needed to investigate the effects of semen incubation or other semen manipulations on the sex ratio of cattle.

During the last half of the 20th century, the world pork industry has achieved astonishing developments in pig breeding. Now swine farms are larger, ownership more concentrated, and farms have become more industrialized. Artificial insemination (AI) plays an important role in animal breeding increasing utilization of genetically superior sires. Currently boars selected for commercial use as AI sires are evaluated on grow-finish performance and carcass characteristics. The objectives of this study were to (A) estimate genetic correlations between production and semen traits in the boar; average daily gain (ADG), back fat thickness (BF) and muscle depth (MD) as production traits, and total sperm cells (TSC), total concentration (TC), volume collected (SV), number of extended doses (ND), and acceptance rate of ejaculates (AR) as semen traits; (B) to model the variances and covariances of total sperm cells (x 10⁹) over the active lifetime of AI boars; and (C) to compare multiple traits and random regression analyses applied to total sperm cells (TSC). Average heritability estimates were 0.39 for ADG, 0.32 for BF, 0.15 for MD, and repeatability estimates were 0.38 for SV, 0.37 for TSC, 0.09 for TC, 0.39 for ND, and 0.16 for AR. Semen traits showed negative genetic correlations with MD. Genetic correlations would indicate that current selection objectives are having a negative effect on semen traits. Therefore, current AI boar selection practices may be having a detrimental effect on semen production. In random regression analysis for total sperm cells, maximum log likelihood value was observed for sixth, fifth, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Best fit as determined by Akaike's Information Criterion was based on a model with sixth, fourth, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Best fit as determined by Schwarz Criterion was by fitting fourth, second, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Heritability estimates for total sperm cells ranged from 0.27 to 0.61 across age of boar classifications. Heritability for total sperm cells tended to increase with age of boar classification. The cyclic nature of heritability for total sperm cells that was observed over the active lifetime of boars may be due in part to number of observations across seasons limiting our ability to correct for seasonal effects on sperm production. In MTDFREML analysis, heritability estimates of 9, 12, 15, 18, 21, 24, and 27 months of age were, respectively, 0.28, 0.29, 0.26, 0.27, 0.30, 0.79, and 0.41. The results from MTDFREML seemed to be overestimated when compared to random regression. Therefore, random regression methods are the most appropriate to analyze semen traits as they are longitudinal data measured over the boars lifetime.

This thesis is aimed at improving techniques for induced maturation and artificial fertilization of Morone. Chapter 1 is a study of hybrid bass (n = 73) exposed to 6- or 9-month photothermal cycles which were formed by excising summer or summer and fall photothermal conditions from an annual regime. Shortened cycles abbreviated the interval between spawning and recrudescence, and advanced maturation by five and four months, respectively. All females developed functionally mature oocytes and ovulated after hormone injection. Maximum testicular sizes were similar and occurred after similar periods (153-199 d). Milt was produced in most (> 90%) males. Some milt characteristics differed among males in the three cycles, but fertility was similar. Findings suggest that excised cycles can advance maturation in hybrid bass, permitting off-season spawning. In Chapter 2, effects of stripping frequency (3 times in 7 days) on semen quality was determined by measuring concentration, motility, osmolality, pH and sugars of milt. Male and female effects on fertility variation were measured by crossing all males and females. Sperm concentration was estimated by spermatocrit, ultraviolet and fluorometric absorbance. Motility (58 sec) and milt pH (8.44) were lowest (p < 0.05) after the third stripping. Concentration (1.76 x 10⁹ cells mL⁻¹) and osmolality also decreased with stripping frequency, but inconsistently among males. Fertility was highest during the second stripping and was correlated with milt pH (r = 0.762). Fertility variation was caused by females (p = 0.001). Results provide the first characterization of Morone semen quality. They suggest that frequent stripping alters milt parameters, that females account for most fertility variation, and that sperm concentration can be estimated reliably by spermatocrit.

Studies presented in this thesis investigated alterative extender-cryoprotectant combinations for semen preservation in red deer (Cervus elaphus) and fallow deer (Dama dama). Semen was collected by electroejaculation under anaesthesia and pooled from four red deer stags and four fallow deer bucks. The ejaculated semen was diluted to a final concentration of 100x10⁶ sperm/ml in sodium citrate, tris citrate, skim milk, lactose and ram synthetic based extenders, with glycerol, propan-1, 2-diol (PROH) and dimethyl sulphoxide (DMSO) cryoprotectants at two concentrations, undiluted (U) and half dilution (0.5D). Semen was frozen in 0.25 ml straws in an automated chamber freezer and stored immersed in liquid nitrogen. In vitro laboratory evaluation of frozen-thawed semen involved the recording of motility and progressive motility of sperm by phase contrast microscopy over a 4 hour incubation period at the equivalent rectal body temperature of female red and fallow deer. Post-thaw (t=0) acrosomal status and sperm morphology were determined by histology using phase contrast optics. Red deer semen gave the highest post-thaw motility (56.7±8,4%) and incubation performance with tris citrate-glycerol (0.5D) extender. Glycerol was the overall optimum cryoprotectant at the undiluted concentration for both deer species, the above being the only exception. Lactose glycerol (U) was the superior extender buffer (p0.10), with a genotype effect (P

In a preliminary study of seasonal reproductive changes in stallions, four blood samples per week were collected on three separate days from each of four standardbred and two pony stallions for ten consecutive months. Each serum sample was analysed for testosterone, LH and FSH using radio-immunoassay techniques. During the same period paired ejaculates were collected one hour apart one day per week and volumes of both gel-free semen and gel, motility, spermatozoa/ml, total spermatozoa/ejaculate and pH were measured for each semen sample. Libido was monitored by recording reaction time and number of mounts/ejaculate for each collection. On the days of semen collection two blood samples were taken, one before sexual stimulation and one immediately after the first ejaculate. There was a marked effect of season on serum testosterone, LH and FSH levels, however the extent of change and timing of peak levels varies between stallions. Serum FSH levels started rising in late July-early August and generally reached a sharp peak during September, followed by a gradual decline thereafter. In several cases, testosterone showed a pattern of change similar to the of FSH, with peak levels occurring 2-4 weeks after FSH. This apparent association between these two hormones suggested that FSH may modify testosterone production possibly by altering the responsiveness to the testes to LH stimulation. In contrast, although LH levels began to rise at about the same time as FSH levels, peak values were not reached until November, two months after FSH peaked. The seasonal pattern of circulating LH levels more closely resembled the annual changes in daily photoperiod. These results, along with seasonal changes in seminal parameters, were consistent with the view that FSH is involved in initiating early seasonal changes in spermatogenic activity. There was some evidence for an inverse relationship between testicular function and FSH levels which supported the concept of a negative feedback on FSH secretion by a principle of testicular origin. Only LH was significantly (P

วัตถุประสงค์: เพื่อศึกษาปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิของผู้ใหญ่ชายไทยในการใช้เป็นข้อมูลอ้างอิงของประเทศไทย เพื่อศึกษาความสัมพันธ์ระหว่างปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิกับอายุประชากร และเพื่อศึกษาความสัมพันธ์ระหว่างปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิกับปริมาณน้ำอสุจิที่หลั่งออกมาในแต่ละครั้ง วิธีการศึกษา: เป็นการวิจัยเชิงทดลอง โดยใช้ตัวอย่างน้ำอสุจิที่ได้รับบริจาคจากอาสาสมัครที่ลงนามในหนังสือให้ความยินยอมในการเข้าร่วมการวิจัยที่ผ่านการรับรองจากคณะกรรมการจริยธรรมการวิจัยในคน คณะแพทยศาสตร์ศิริราชพยาบาล มหาวิทยาลัยมหิดล อาสาสมัครทุกรายเป็นเพศชาย สัญชาติไทย มีอายุ 20 - 60 ปี มีสุขภาพดี ไม่มีไข้ภายใน 5 วันก่อนและขณะเก็บตัวอย่าง และได้รับการร้องขอให้ปฏิบัติตามเกณฑ์คัดเข้าอย่างเคร่งครัด คือ งดเพศสัมพันธ์หรือการสำเร็จความใครด้วยตัวเองอย่างน้อย 5 วันก่อนเก็บตัวอย่าง ตัวอย่างน้ำอสุจิทั้งหมดถูกเก็บไว้ที่อุณหภูมิ -80 องศาเซลเซียส ก่อนนำไปตรวจหาปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากด้วยวิธี enzyme-linked immunosorbent assay (ELISA) ตามวิธีที่ผู้วิจัยได้รายงานไว้แล้ว ผลการศึกษา: ตัวอย่างน้ำอสุจิที่ใช้ในการศึกษาได้จากอาสาสมัครชายไทย จำนวน 17 คน อายุเฉลี่ย 32.9 gif.latex?\pm 10.2 ปี (22 - 55 ปี) ปริมาณน้ำอสุจิเฉลี่ย 2.5 gif.latex?\pm 0.6 มิลลิลิตร (1.5 - 3.5 มิลลิตร) จากการศึกษานี้พบว่าระดับแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิมีค่าอยู่ระหว่าง 503,636 - 519,090 นาโนกรัมต่อมิลลิลิตร โดยมีค่าเฉลี่ย 513,502 gif.latex?\pm 4,211 นาโนกรัมต่อมิลลิลิตร และพบว่าปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิมีความสัมพันธ์อย่างผกผันกับอายุของประชากร (r = -0.547, P < 0.01) สรุป: จากการศึกษานี้ผู้วิจัยได้รายงานปริมาณแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิของผู้ใหญ่ชายไทย และพบว่าแอนติเจนที่จำเพาะต่อต่อมลูกหมากในน้ำอสุจิมีปริมาณน้อยลงเมื่อประชากรตัวอย่างมีอายุมากขึ้นและมีปริมาณเพิ่มขึ้นเมื่อปริมาณน้ำอสุจิที่หลั่งออกมาในแต่ละครั้งมากขึ้น อย่างไรก็ตามอาจมีความจำเป็นในการศึกษาเพิ่มเติมด้วยจำนวนตัวอย่างที่มากขึ้นเพื่อยืนยันผลการศึกษานี้