Semen Doses Research Articles

117 Evaluation of Semen Production and Biomarkers Produced by Boars Fed Microfused Essential Oil Technology in Their Diet

Abstract Two experiments were conducted to evaluate the effect of feeding Microfused Essential Oil Technology (MEOT, Ralco Nutrition, Inc.) to mature boars on their semen production and semen quality biomarkers. In Experiment 1, boars (n = 21) housed at a commercial boar stud were fed MEOT in their diet. These boars were collected in their normal collection routine of twice per week. Total motile sperm count was measured for each collection over a 10-month period (December to September). A baseline motile sperm count was determined for each boar during the 2 months before the beginning of the MEOT feeding in February. Repeated measures analysis was conducted to evaluate the effects of sperm production over time. Feeding MEOT to boars resulted in greater motile sperm count (P < 0.0001) and greater (P < 0.0001) economic revenue per collection. In Exp. 2, the objective was to determine if the increased semen production measured had any impact on semen quality biomarkers. Semen samples were collected from the same boar stud used in Exp. 1 after MEOT feeding. Control samples were collected from a boar stud using similar boar genotypes, environmental condition, semen extender, and time from collection. One 75 mL extended semen dose from 8 to 10 boars across 3 weeks was collected and sent to the laboratory for semen quality analysis. The traits and biomarkers that were analyzed for each sample included morphology, zinc signature, plasma membrane integrity (detected by propidium iodide; PI), lectin PNA acrosome health, lectin LCA membrane health, reactive oxygen species status (ROS; detected by H2DCFDA), aggresome (AGG), and DNA fragmentation (dected by acridine orange). No differences in morphology (P > 0.05) were detected. For the MEOT-fed boars, a greater portion (P < 0.001) of the sperm population was in the zinc signature 2 category indicating more live, non-capacitated sperm and a lesser portion (P < 0.0003) in the zinc signature 4 category indicating less dead, fully capacitated sperm. In addition, for PI, a greater portion (P < 0.03) of the sperm were live with good plasma membrane integrity for boars fed MEOT versus the controls. A greater portion of the sperm from the MEOT-fed boars had better acrosome integrity (P < 0.001) as well as tail membrane health (P < 0.01). No differences (P > 0.05) were observed for ROS. Semen from MEOT-fed boars had less AGG misfolded proteins (P < 0.0001) and a tendency (P < 0.07) for less DNA fragmentation. These data indicate that feeding MEOT to boars resulted in greater motile sperm production and that did not result in a negative impact on semen quality with many semen biomarkers being improved in MEOT-fed boars.

60 Molecular Characterization of Boar Semen in Relation to Sperm Quality

Abstract Artificial insemination (AI) with fresh semen is the primary breeding tool in the swine industry. However, the heterogenous semen doses create challenges in boar studs due to the lack of consistent assessment criteria. Seminal plasma (SP) surrounding spermatozoa is a great source of reliable non-invasive biomarkers of sperm quality. It contains a wide array of bioactive molecules, whose dynamic compositions in response to extrinsic and intrinsic factors influence sperm motility and morphology. Therefore, we hypothesize that the miRNA content of extracellular vesicles (EVs) found in SP may reflect semen quality. Our study aimed to characterize poor vs. good quality semen and compare miRNA profiles of SP-derived EVs. Raw semen was collected (n = 75) from sexually mature Duroc boars at a commercial stud (Prestage Farms, MS) for 8 weeks. Semen samples were subjected to sperm motility and morphology analyses and subsequently classified as poor (n = 38) or good (n = 37) quality based on assessment criteria cut-offs (< 70% and ≥70%, respectively). Thereafter, raw semen samples were subjected to serial centrifugation to collect spermatozoa and SP. Samples were stored at -80oC until analyses. A subset of each frozen-thawed SP was used for NIRS absorbance analysis (ASD FieldSpec3 portable spectrometer – 1mm quartz cuvettes; 750-2500 nm). Other subsets of frozen-thawed SP were ultra-centrifugated to isolate EVs (SP-EV), followed by confirmations through transmission electron microscopy (TEM), dynamic light scattering (DLS), and immuno-western blotting. Five pools of three individual SP-EV samples, corresponding to extremely poor and extremely good quality semen, were constituted for total exosome RNA isolation, small RNA sequencing, miRNA detection, and differential miRNA expression. Messenger RNA targets of miRNA were searched and subjected to functional bioinformatic analyses using various software algorithms. Significant differences were set for P < 0.05. Poor and good quality samples had different spectrum profiles following NIRS analyses. Changes were explicitly observed in water structures at coordinates C1, C5, and C12 (nm), corresponding to water associations with chaotropic solutes (water asymmetrical stretching vibrations-ν3), free water, and kosmotropic solutes (increased H-bonding numbers and strength), respectively. The NIRS-LDA data contrasted two distinctive poor and good quality SP samples. All boar SP-EV were confirmed: round spherical structure; 30 to 400nm of diameter; and CD9+, CD63+, and CD81+ membrane markers. Totals of 281 and 271 miRNAs were identified in poor and good groups, respectively, and 15 were differentially expressed (DE). Only three DE miRNAs (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) permitted targeting of 109 mRNA, which functional analyses indicated associations with nuclear and cytosol localization, as well as with functions, i.e., acetylation, Ubl conjugation, protein kinase binding, potentially altering sperm function. In conclusion, the study indicates that the NIRS tool can distinguish between good and poor semen quality, and the miRNA SP-EV content may reflect semen quality.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Ram semen quality after supplementation with gelatin, agar or alginate prior to cooling storage.

Adding gelling agents to convert the liquid state of the semen extender to a solid state allows for an increased sperm life span. Gelatin and alginate have been used to study the effects of gelling agents on sperm quality. However, there are other gelling agents that have not been studied, such as agar. In addition, studying different sources of gelling agents or the effect of mixing more than one gelling agent with semen extenders on sperm fertility has received little attention. Therefore, the objective of this study was to evaluate the effect of adding agar and a mixture of gelling agents from different sources to semen extender on ram sperm traits and fertility. The first trial evaluated the effect of the addition of 2.5-3 mg mL-1 of gelatin mixed with 0.5-20 mg mL-1 of agar or alginate to ram semen extender on sperm (motility, progressive motility, live/dead, membrane integrity) and semen (pH) characteristics. The response variables were evaluated 1, 72 and 144 h after storage at 4°C. In the second trial, two sources (feed grade and bacteriological) of gelatin and agar were evaluated on the response variables as in Trial 1. In trial 3, a total of 34 ewes were inseminated with doses supplemented (n = 17) with or without (n = 17) agar and gelatin. The pregnancy rate was diagnosed 40 days after insemination. In general, adding agar and gelatin improves (p < .05) sperm motility, membrane integrity and the ratio of live sperm after 144 h of storage compared to the Control group, regardless of the source (bacteriological or feed grade). However, the pregnancy rate in ewes was not influenced (p ≥ .05) by semen doses stored with agar and gelatin. In conclusion, the addition of agar and gelatin preserves ram sperm motility and membrane integrity after 144 of storage at 4°C without affecting the pregnancy rate in inseminated ewes.

Sperm concentration of boar semen doses and sperm quality: Novel perspectives based on the extender type and sperm resilience

This study evaluated the effect of sperm concentration of boar semen doses on their capacity to maintain its motility over the thermo-resistance test (TRT; sperm resilience) and verified if the extender type (short or long-term) could influence this effect. Thirty ejaculates from five crossbred mature PIC® boars were used, and a factorial design was followed to produce semen doses with 1.5 billion cells in 45 or 90 mL, using Beltsville Thawing Solution (BTS) or Androstar® Plus (APlus). Then, low-concentration doses (16.7 × 106 cells/mL in 90 mL) and higher-concentration doses (33.3 × 106 cells/mL in 45 mL) with BTS or APlus were produced and stored at 17 °C for 168 h. At 72 h, during the TRT, the low-concentration doses (16.7 × 106 cells/mL) lost three-fold less motility than doses with 33.3 × 106 cells/mL (P < 0.01), regardless of the extender type (11. 5% vs. 30.5% of initial motility, respectively). Similar results were found when the TRT was carried out at 168 h, with low-concentration doses losing two-fold less motility (11.4%) than highly concentrated doses (25.9%; P < 0.01). No sperm concentration effect was observed on membrane integrity or mitochondrial membrane potential (P ≥ 0.23). The osmolarity was not affected by the sperm concentration (P = 0.56), only by the extender and the storage time (P < 0.01). In conclusion, the sperm concentration effect on sperm quality was not influenced by extender type, and the data suggest that a low concentration of semen doses positively affects sperm resilience.

Microbial composition of goat buck's ejaculates is modified by the process of preparing and storing refrigerated semen doses

Ejaculates present their own microbiota, and a link between ejaculates' microbiota and sperm quality and fertility exists. With the development of artificial insemination in animal breeding, ejaculates must be manipulated by diluting them with extenders and storing them at temperatures below body temperature. The effects that these processes have on the original semen microbiota have never been studied. This study explores the effects of the protocol for preparing refrigerated goat buck semen doses and storing on seminal microbiota. Semen from six adult goat bucks of the Murciano-Granadina breed (24 ejaculates) was used, cooled to 4 °C in a skimmed milk-based extender, and stored at this temperature for 24 h. Samples were taken in different steps: in the raw ejaculates (ejaculates), after dilution with the refrigeration extender (diluted), immediately after reaching 4 °C (chilled 0 h) and the samples refrigerated at 4 °C and stored at this temperature for 24 h (chilled 24 h). Sperm quality (motility and integrity of plasma and acrosomal membrane, and mitochondrial functionality) was also evaluated. Bacterial 16S rRNA sequencing was used to study the seminal microbiota. Our results indicated that both refrigeration and storage at 4 °C negatively affected sperm quality parameters. Preparing semen doses and their subsequent conservation caused a significant change in the bacterial community structure. Raw ejaculates showed a lower Pielou's evenness index than the other samples (diluted, chilled 0 h and chilled 24 h). Ejaculates also had a lower Shannon's diversity index (3.44) than the diluted semen (4.17) and the semen chilled for 24 h (4.43). Regarding beta diversity, significant differences were detected between ejaculates and the other treatments. Differences were also found in unweighted UniFrac distances between the semen chilled for 0 h and that chilled for 24 h. At the genus level, marked effects of preparing doses and their subsequent conservation were also evident: 199 genera that were absent in ejaculates were found in the semen chilled and stored for 24 h; 177 genera that were present in ejaculates disappeared after 24-h refrigeration. In conclusion, the extender and protocol for preparing refrigerated goat buck semen doses considerably modify microbial ejaculate composition.

Colloid centrifugation as an alternative to antibiotics in stallion semen preparation

Antibiotics are added to semen extenders to inhibit bacterial growth and maintain sperm quality in semen doses during storage and transportation. The low concentration of antibiotics used, however, could contribute to increased antimicrobial resistance. Recent studies with boar semen suggest that centrifugation through a low density colloid could separate the spermatozoa from most of the bacteria. Such methods can only be recommended for stallion semen if they are without detrimental effects on sperm characteristics. Therefore, the purpose of this pilot study was to investigate if semen processing by single layer centrifugation through a low density colloid reduces the bacterial load of stallion semen without impairing sperm characteristics. Six healthy and fertile stallions were available at the Centre for Artificial Insemination of the University of Veterinary Medicine Vienna. One ejaculate from each stallion was collected and divided into three parts. Each part was either extended to 15 mL (100 million/mL) and processed by single layer centrifugation with 15 mL of 25% low density colloid (SLC), extended with Equiplus without antibiotics (Minitube, Tiefenbach, Germany), or kept as unprocessed control (raw semen). Aliquots from all samples were sent to the Institute of Microbiology for bacteriologicalexamination where they were serially diluted, plated onto blood agar plates, and incubated under aerobic and anaerobic conditions. The number of bacterial colony forming units (cfu/mL) was determined after 4 days of incubation. Sperm motility and viability in SLC and extended samples were evaluated by computer assisted sperm analysis (CASA; SpermVision, Minitube). The SLC and extended samples were stored at 5°C and re-evaluated after 24 h. For statistical analysis, the mean number of bacterial cfus and sperm characteristics were compared among treatments by one-way ANOVA and Tukey's test, with significance set at p < 0.05. Overall sperm yield after SLC was 76.6% (±16.4). Bacterial cfus were lower in SLC samples and extended samples compared to raw semen (4.5 × 106 cfu/mL, (14.5 × 106cfu/mL, and 8.3 × 106, respectively; p= 0.017, and p = 0.038, respectively). Sperm motility was higher in SLC samples at both time points (93.8% at 0 h, p = 0.001; 87.7% at 24h, p = 0.007) than in extended semen at 24 h (56.3%). Sperm viability was higher in the SLC at both time points (88.4% at 0 h, p = 0.0002; 86.4% at 24h, p = 0.002) than in extended semen at 24 h (73.2%). In conclusion, bacterial load, and sperm characteristics, were improved in SLC compared to extended stallion semen. An extended investigation is warranted.

Quality of frozen equine semen stored in different shipping containers

The use of cryopreserved semen in horses requires sophisticated equipment, including containers maintaining the temperature of liquid nitrogen (LN). Simpler and less expensive devices for transport of frozen semen may help breeding programs in developing countries and reduce the costly return of empty containers. Veterinarians sometimes question the quality of frozen-thawed shipped semen because of potential fluctuations in shipping temperatures. This study aimed to investigate the effects of shipping duration and temperature on post-thaw quality of frozen semen stored either in a dry shipper (≤ -150°C) or on dry ice (≤ -78°C) for 14 days. Straws (n=165) containing cryopreserved semen from five stallions stored in liquid nitrogen were transferred (D0) to a dry shipper (DS) and to a styrofoam box with dry ice (DI). Straws from DS and DI were returned to LN on D1, D3, D7, D10, and D14. All semen doses were evaluated at each time point by CASA for total (TMot) and progressive motility (PMot), and kinematic parameters such as average path velocity (VAP), curve-linear velocity (VCL) and straight-line velocity (VSL), by fluorescence microscopy for the percentage of membrane-intact spermatozoa (SYBR14/PI stain), and high mitochondrial membrane potential (HMMP; JC-1 stain). Data were analyzed by repeated measures ANOVA with both storage time and storage device as within subject factors. Thetemperature inside the DS and DI containers was monitored continuously with a Tempod 200XB data logger. There were no temperature fluctuations during the 14-day storage in both containers. Until D7 of storage, no changes were observed in TMot, PMot, and membrane-intact spermatozoa (%). Thereafter all three parameters decreased in semen stored in DI but not in DS (storage device at least p<0.05, time p<0.01, time x storage device p<0.001; e.g., TMot D7 on DI and in the DS both 59±14 versus 34±10 and 60±13%, respectively, on D14; membrane-intact spermatozoa D7 on DI 50±13 and in the DS 52±12 versus 28±7 and 53±19% on D14). The HMMP percent decreased continuously during storage in both containers (p<0.001) and this decrease was more pronounced for DI compared to DS (time x storage device p<0.05; DI from 43±4 on D1 to 26±3% on D14, DS from 43±5 on D1 to 30±4% on D14). All velocity parameters remained constant with storage in both devices. In conclusion, cryopreserved frozen stallion semen can be stored for up to 7 days on dry ice without negative effects on post-thaw characteristics. Dry ice may thus be an alternative to liquid nitrogen containers for the short-term transport of frozen stallion semen. Controlled insemination trials to confirm the fertility of frozen stallion semen shipped on dry ice are justified.

An alternative method to circumvent poor semen cooling and fertility of stallions

Cooled-shipped semen is routinely used in the equine breeding industry. Although most stallions present satisfactory semen quality and fertility after cooling and transportation, a population of stallions offer a suboptimal sperm cooling tolerance even when strategies to enhance stallion semen cooling are applied. Therefore, it is critical to developing alternative methods to improve the post-cooling semen quality from this population of stallions. This study assessed sperm parameters and fertility of cooled-stored stallion semen processed and resuspended in three extenders. In experiment 1, semen was collected from 21 stallions classified as having good (“Good coolers,” n=8) or poor (“Bad coolers,” n=13) sperm characteristics during cooled-storage after semen dilution in milk-based extender (SM, BotuSemen®). Immediately after semen collection, semen was extended at 50 million/mL in SM extender and stored at 5°C for 24h. Thereafter, cooled-stored semen was processed by SpermFilter® or conventional centrifugation (600 × g/10 min), and the resulting pellets were resuspended in SM, SM containing pentoxifylline (SM-P, BotuSemen Turbo®), or egg yolk-based extender containing cryoprotectants (EY, BotuCrio®). Unprocessed cooled semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. In experiment 2, 66 cooled semen doses from stallions (n=9) classified as Bad coolers were randomly used to inseminate18 mares in 66 cycles (Unprocessed, n=22; SpermFilter®/SM-P, n=16; or SpermFilter®/EY, n=28 cycles). Data were analyzed with a mixed model, Tukey's, and logistic regression. Overall, resuspending centrifuged (Total motility [TM], 73±14%; progressive motility [PM], 36±13%) or filtered (TM, 75±15%; PM, 37±13%) semen in EY enhanced (P<0.05) sperm motility compared to unprocessed semen (TM, 50±33%; PM, 23±18%) and the other groups. However, when semen from only Bad Coolers was analyzed, motility of semen processed by SpermFilter® and resuspended in SM-P was enhanced (TM, 52±19%; PM, 22±14%) compared to unprocessed semen (TM, 30±26%; PM, 10±10%) and similar (P>0.05) to semen resuspended in EY (TM, 69±16%; PM, 34±14%). Pellet resuspension with EY (52±25%) and SM-P (49±25%) improved the HMMP of Bad cooler stallions (P<0.05) compared to unprocessed semen. Semen processed by SpermFilter® had superior PMI (72±8%) than centrifuged semen (64±11%; P<0.05) overall. In experiment 2, pregnancy rate of mares inseminated with SpermFilter®/SM-P (50%) or SpermFilter®/-EY (68%) was greater than with unprocessed semen (14%) (P<0.05). In conclusion, semen parameters and fertility of stallions with poor semen cooling can be enhanced by SpermFilter® and resuspension of the pellets with EY or SM-P. Acknowledgments: The authors thank FAPESP (grant #2017/13883-9) for the financial support.

Novel interpretation of sperm stress test and morphology for maturity assessment of young Norwegian Red bulls

The use of genomic selection significantly reduces the age of dairy bulls entering semen production compared to progeny testing. The study aimed to identify early indicators that could be used for screening bulls during their performance testing period and could give us insight into their future semen production performance, acceptance for the AI station, and prediction of their future fertility. The study population consisted of 142 young Norwegian Red bulls enrolled at the performance test station, followed until we received semen production data, semen doses, and, subsequently, non-return rates (NR56) from the AI station. A range of semen quality parameters were measured with computer-assisted sperm analysis and flow cytometry from ejaculates collected from 65 bulls (9–13 months). The population morphometry of normal spermatozoa was examined, showing that Norwegian Red bulls at 10 months of age have homogenous sperm morphometry. Norwegian Red bulls could be separated into 3 clusters according to their sperm's reaction patterns to stress test and cryopreservation. Results of semi-automated morphology assessment of young Norwegian Red bulls showed that 42% of bulls rejected for the AI station and 18% of bulls accepted had ejaculates with abnormal morphology scores. For the youngest age group at 10 months, the mean (SD) proportion of spermatozoa with normal morphology was 77.5% (10.6). Using novel interpretation of sperm stress test combined with sperm morphology analysis and consecutive cryopreservation at a young age allowed identification of the candidate's sperm quality status. This could help breeding companies introduce young bulls earlier to the AI stations.

Bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage with or without antibiotics in a tropical environment

BackgroundIn tropical environments, boar semen is prepared either from a boar on the same farm as the sow herd or collected in semen collection centers and then transported to other farms. Thus, the semen doses can be used for artificial insemination either immediately or preserved for 2–3 days. The present study investigated the bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage in semen extender with or without antibiotics in Thailand.M&MIn total, 20 Duroc ejaculates were collected. Each ejaculate was diluted in Beltsville Thawing Solution extender either with 0.25 g of gentamicin per liter (ANTIBIOTIC) or without gentamicin (NO-ANITIBIOTIC) to create semen doses containing 3,000 × 106 sperm/100 mL. These were stored at 17 °C for 4 days. Semen characteristics and total bacterial count (CFU per mL, log10) were measured after collection and during storage.ResultsSperm viability was decreased by 6.4% for every 1.0 log10 increase in total bacterial count (p = 0.026) and Staphylococcus spp. were the most frequently isolated across ejaculates. Throughout the 4 days of storage, sperm motility, viability and acrosome integrity in the ANTIBIOTIC group were higher than those in the NO-ANTIBIOTIC group (p < 0.05), while the total bacterial count was lower (1.9 ± 0.1 versus 3.9 ± 0.1 log10, respectively; p < 0.001). Without antibiotic supplementation, the total numbers of bacteria counted on days 2 and 3 of storage were higher than those determined on days 0 and 1 (p < 0.001). Differences in semen quality were detected on days 2 and 3 between the NO-ANTIBIOTIC and ANTIBIOTIC groups in high-viability semen (p < 0.05). However, no differences in sperm quality between the NO-ANTIBIOTIC and ANTIBIOTIC groups were detected in the low-viability semen on each storage day (p > 0.05). On the last day of preservation, Globicatella sanguinis (57.2%), Delftia acidovorans (18.9%) and Micrococcus spp. (5.9%) remained as the top three most abundant contaminants in the semen with antibiotic.ConclusionOur findings contribute new insights toward reducing antibiotics as well as rational antibiotic use in the boar AI industry. The growth of bacteria was significantly greater only after 2 days of preservation in the semen without antibiotic. For semen doses diluted from highly viable ejaculates, it is possible to store for 2 days without any antibiotic supplementation. Moreover, bacterial counts increased at the end of storage in the presence of gentamycin, suggesting the loss of bacteriostatic properties of gentamicin to the growth of bacteria during storage.

Evaluation of the Effects of Melatonin Implants on Testicular and Spermatic Parameters of Rams Raised in Station in the Niayes Zone in Senegal

The intensification of livestock production in response to the growing demand for animal products is increasingly creating new challenges in terms of control of livestock management and feeding, and especially reproduction. Also, ewes are often considered responsible for infertility problems in a sheep flock, as the reproductive capacity of rams is often taken for granted. However, it is important to have genetically superior males to improve the overall fertility of the herd. And, one way to control the sexual performance of rams and improve their fertilizing capacity, is to have them undergo melatonin treatment. The objective of this study was to evaluate the effects of the use of melatonin implants on testicular and spermatic parameters of rams raised in stations in the Niayes area of Senegal. The experimental herd consisted of 6 rams of well-known body characteristics which were followed for 2 months during which testicular measurements were carried out at the beginning and semen was collected by electroejaculation every 2 weeks. After a break of 2 weeks, the same rams are implanted with melatonin (MELOVINE ®) at a rate of 3 implants/ram, i.e. a dose of 54 mg of melatonin per ram, and the same measurements and semen collections are again carried out for 2 months. Data on antero-posterior testicular diameter, testicular weight, scrotal circumference, and semen analysis (volume, color, appearance, mass and individual motility, concentration, morphology) recorded and processed with Excel were exported to the SPSS software where they were subjected to descriptive statistical analysis and independent sample comparison testing. The results showed that melatonin treatment significantly reduced weight (555±155 g vs. 396±128 g) and antero-posterior diameter (23.42±2.0 cm vs. 20.10±2.26 cm). However, it induced improvements in ejaculate volume, color, and appearance, sperm production, and morphological abnormality rate, and, most significantly, sperm motility (2.1±1.5 vs. 3.3±1.2) and survival rate (54±34% vs. 80±31%) of spermatozoa. Melatonin has therefore improved the sperm characteristics of rams, and is an interesting reproductive management tool for improving the productivity of sheep farms. It can be recommended for use in artificial insemination centres in order to improve the characteristics of the semen doses produced.

27 Bacterial Community Diversity in Fresh Extended Boar Semen

Abstract Status of bacterial diversity within the male seminal microbiome has previously been linked to fertility status in mammals. However, minimal research has focused on the reproductive microbiome in boar semen. The objective of this study was to analyze the diversity of bacterial communities within fresh extended boar semen samples 1) between studs, 2) over 5 consecutive days, and 3) between pooled and single-sire doses. Eight single-sire (n = 4 per stud) and eight pooled (n = 4 per stud) fresh extended semen doses (80 mL) were obtained from two boar studs (A vs. B). Pooled doses were the composite of boars in single-sire doses. Doses were subsampled (16 mL) for 5 days post-collection. Negative controls remained unopened until the last day. Each day, 5 mL of each subsample were flash frozen and stored at -80°C for bacterial analysis targeting the V4 hypervariable region of the 16S rRNA gene for Illumina sequencing. Quality of all sequences was determined by FastQC before being processed for taxonomic analysis using the qiime2 pipeline. Alpha diversity (difference within sample metric) was analyzed using Kruskal-Wallis test, whereas PERMANOVA was performed for beta diversity analysis (differences between sample metric). According to alpha diversity metrics, Chao1 (P &amp;lt; 0.01) and Shannon’s index (P &amp;lt; 0.01) indicated contrasts in diversity among all days and the negative controls. This was further confirmed with a pairwise comparison between individual groups, in which day 1 exhibited lower alpha diversity when compared with days 2, 3, 4, and 5 using Chao1 (P &amp;lt; 0.01) and Shannon’s index (P &amp;lt; 0.01). Interestingly, reduced diversity was observed for day 1 compared with the negative controls with Chao1 (57.4 ± 6.6 vs. 132.4 ± 22.1, P &amp;lt; 0.01, Q &amp;lt; 0.01; respectively) and Shannon’s index (4.8 ± 0.2 vs. 6.1 ± 0.3, P &amp;lt; 0.01, Q &amp;lt; 0.01; respectively). No differences between boar stud or sire-type were detected for alpha diversity metrics. For beta diversity analyses, both PCoA plots and PERMANOVA for weighted unifrac metric demonstrated no significant clustering between sampling days. However, distinctions between all days and negative controls were identified using PERMANOVA for unweighted unifrac metric. Specifically, differences were discovered between day 1 and days 3, 4, and 5 (P &amp;lt; 0.01, Q &amp;lt; 0.01). PERMANOVA for both weighted (P &amp;lt; 0.01) and unweighted (P &amp;lt; 0.01) unifrac metrics revealed distinct clustering by boar stud. However, there were no differences in beta diversity between pooled and single-sire samples for both weighted and unweighted unifrac metrics. Based on these results, bacterial diversity within a semen sample may be altered over time and could occur between boar stud operations. Additional studies analyzing the reproductive microbiome of semen could help improve the characterization of fertility status in boars.

024 Resulting sex ratio and pregnancy success utilizing different doses of sex-sorted heterospermic semen in beef cows submitted to fixed-time artificial insemination

024 Resulting sex ratio and pregnancy success utilizing different doses of sex-sorted heterospermic semen in beef cows submitted to fixed-time artificial insemination

Spermatozoa cooled to 5°C oneday after collection from porcine commercial semen doses retain sperm functionality with reduced bacterial load.

Commercial porcine semen is stored at 17°C, leading to a reduction of sperm quality and increase of bacterial growth. To evaluate the effect of 5°C storage on porcine sperm functionality cooled one day after collection. Semen doses (n=40) were transported at 17°C and cooled at 5°C one day after collection. Spermatozoa were evaluated at Days 1, 4, and 7 for motility, viability, acrosome integrity, membrane stability, intracellular zinc, oxidative stress, and bacterial growth. Contaminated semen doses predominantly exhibited Serratia marcescens, with increasing bacterial load during 17°C storage. Under hypothermal storage, negative doses for bacteria growth at Day 1 remained negative, and bacterial load did not increase in bacterial contaminated samples. Motility was significantly reduced through 17°C storage, but at 5°C, motility was only reduced at Day 4. Samples with bacterial growth (35.0%, 14/40) had significantly reduced motility at 17°C, but motility was unaltered at 5°C. Plasma membrane and acrosome integrity without bacterial contamination were unaffected at 17°C, but were significantly reduced at 5°C on Day 7. Plasma membrane and acrosome integrity significantly decreased with bacterial contamination regardless of temperature. High mitochondrial activity in viable spermatozoa without bacteria was not altered by temperature, but was significantly reduced by bacterial contamination at 17°C. Membrane stability was significantly reduced at Day 4, but tended (p=0.07) to be higher in samples without bacterial growth. Viable spermatozoa exhibiting high zinc were significantly reduced throughout storage regardless of temperature. Oxidative stress levels were not altered, but significantly increased with bacterial contamination at 17°C. Porcine spermatozoa cooled to 5°C one day after collection retain functional attributes similar to spermatozoa stored at 17°C, but have a reduced bacterial load. Cooling extended boar semen to 5°C is feasible after transport to avoid modifying semen production.

Characterization of the Germplasm Bank for the Spanish Autochthonous Bull Breed "Asturiana de la Montaña".

Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm's viability. This study reports an investigation of the "Asturiana de la Montaña" breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4-6 mL, 5-10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26-43%, progressive 14-28%), with higher values for viability (47-62%). Insemination results showed a good performance for this breed (NRR: 47-56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results.

Effect of various pH levels on the sperm kinematic parameters of boars

The objective of this work was to identify the effects of pH on the structure of kinematic sperm subpopulations using commercial doses of boar semen diluted in Androstar Plus extender. We adjusted the pH value of the diluted semen samples to 7.4, 7.6, 7.8, and 8.0, and evaluated motility parameters using an open-source computer-assisted sperm analysis system. We evaluated the dataset of the kinematic parameters of the individual sperm for the effects of pH on the average values overall and on the subpopulations of sperm. To identify these subpopulations, we reduced the dimensionality of the dataset through principal component analysis followed by hierarchical clustering. We then compared the results for the kinematic parameters across the dataset and in the subpopulations with the average of the corresponding control (i.e., the basal pH value). We found that increasing the pH did not affect the proportion of motile sperm but did affect the manner in which they moved, rendering their movement more linear. The progressive motility increased relative to the control at all pH values above 7.1, while the proportion of sperm with low motility increased at pH 7.8. We distinguished three sperm subpopulations. The proportion of sperm in Subpopulation 1 increased at pH values of 7.8 and 8.0, with the sperm being characterized by low velocity and high values for linearity. The sperm in Subpopulation 3, by contrast, were relatively few but showed relatively high curvilinear velocity. In this study, we demonstrated the presence of subpopulations of boar sperm that responded differentially to changes in pH.

Estimation of sperm concentration limits to produce intrauterine insemination doses in swine.

This study evaluated the effect of sperm concentration of boar semen doses, for intrauterine artificial insemination (IUAI), on semen quality and established concentration limits for their production. Twenty ejaculates from four crossbred mature PIC® boars were collected to produce 50 mL semen doses in a split sample, reaching the following sperm concentrations: ~20, 30, 60, and 100 × 106 cells/mL. Doses were produced using Androstar® Plus, stored at 17°C, and evaluated until 120 h of storage. There was a linear decrease in sperm motility as the sperm concentration increased (p linear < .01). The concentration which no longer affected the total and progressive motility was 59 and 55 × 106 cells/mL, respectively (corresponding to 71% and 62%, respectively). The pH linearly decreased as the sperm concentration increased (p < .01); yet, at 72 and 120 h, the parameter dramatically reduced in boar semen doses with 60 and 100 × 106 cells/mL. The percentage of cells with intact plasma and acrosomal membranes or with high mitochondrial membrane potential was not influenced by the sperm concentration (p ≥ .15). In conclusion, sperm motility was negatively affected in highly (60 and 100 × 106 cells/mL) concentrated doses. To achieve suitable sperm motility, boar semen doses may not surpass the sperm concentration of 55 × 106 cells/mL. The effect of low-concentrated boar semen doses on sperm quality still needs to be better evaluated, mainly considering the influence of extender type and thermo-resistance conditions.

Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species

Single-layer centrifugation (SLC) with a low-density colloid is an efficient method for removing contaminating microorganisms from boar semen while recovering most spermatozoa from the original sample. This study tested the performance of this technique, using 50-ml tubes, by spiking commercial semen doses prepared without antibiotics with selected bacterial species followed by storage at 17 °C. The doses were spiked up to 102/ml CFU (colony forming units) of the bacteria Burkholderia ambifaria, Pseudomonas aeruginosa, and Staphylococcus simulans. The semen was processed by SLC (15 ml of sample and 15 ml of colloid) with the colloid Porcicoll at 20% (P20) and 30% (P30), with a spiked control (CTL) and an unspiked control (CTL0), analyzing microbiology and sperm quality on days 0, 3 and 7. SLC completely removed B. ambifaria and S. simulans, considerably reducing P. aeruginosa and overall contamination (especially P30, ∼104 CFU/ml of total contamination on day 7, median). Sperm viability was lower in P20 and P30 samples at day 0, with higher cytoplasmic ROS. Still, results were similar in all groups on day 3 and reversed on day 7, indicating a protective effect of SLC (possibly directly by removal of damaged sperm and indirectly because of lower bacterial contamination). Sperm chromatin was affected by the treatment (lower DNA fragmentation and chromatin decondensation) and storage (higher overall condensation on day 7 as per chromomycin A3 and monobromobimane staining). In conclusion, SLC with low-density colloids can remove most bacteria in a controlled contamination design while potentially improving sperm quality and long-term storage at practical temperatures.

Intensity and Duration of Vibration Emissions during Shipping as Interacting Factors on the Quality of Boar Semen Extended in Beltsville Thawing Solution

Vibration emissions during the transport of boar semen for artificial insemination (AI) affect sperm quality. In the present study, the common influence of the following factors was investigated: vibrations (displacement index (Di) = 0.5 to 6.0), duration of transport (0 to 12 h) and storage time (days 1 to 4). Normospermic ejaculates were collected from 39 fertile Pietrain boars (aged 18.6 ± 4.5 months) and diluted in a one-step procedure with an isothermic (32 °C) BTS (Minitüb) extender (n = 546 samples). Sperm concentration was adjusted to 22 × 106 sperm·mL-1. Extended semen (85 ± 1 mL) was filled into 95 mL QuickTip Flexitubes (Minitüb). For transport simulation on day 0, a laboratory shaker IKA MTS 4 was used. Total sperm motility (TSM) was evaluated on days 1 to 4. Thermo-resistance test (TRT), mitochondrial activity (MITO) and plasma membrane integrity (PMI) were assessed on day 4. Sperm quality dropped with increasing vibration intensity and transport duration, and the effect was enhanced by a longer storage time. A linear regression was performed using a mixed model, accounting for the boar as a random effect. The interaction between Di and transport duration significantly (p < 0.001) explained data for TSM (-0.30 ± 0.03%), TRT (-0.39 ± 0.06%), MITO (-0.45 ± 0.06%) and PMI (-0.43 ± 0.05%). Additionally, TSM decreased by 0.66 ± 0.08% with each day of storage (p < 0.001). It can be concluded that boar semen extended in BTS should be transported carefully. If this is not possible or the semen doses are transported a long way, the storage time should be reduced to a minimum.