Extended Boar Semen Research Articles

Quality and fertility of extended boar semen after prolonged transport

The aim of this study was to analyse semen quality parameters of fresh-extended porcine semen imported from Denmark through prolonged importing regulations and transport and to analyse fertility performance in sows inseminated four days post semen collection. Semen was analysed immediately upon the arrival (4 days post collection) using computer-assisted sperm analyses (CASA), cytomorphology and flow cytometry, and sows were inseminated later the same day. The mean sperm concentration was 1718.09 ± 100.11 × 106 , with average of 1193.40 ± 90.58 × 106 motile cells (69%) and 756.04 ± 70.57 × 106 progressive motile spermatozoa (43.58%) in a dose, which was almost twice lower in value than producer certified-declared number of 2 billion of motile cells. Acrosome and sperm membrane integrity test indicated high percentage of total damaged acrosome. Mean values of semen samples analysed by sperm chromatin structure assay (SCSA) showed relatively high degree of chromatin damage. Membrane permeability test showed high percentage of sperm with damaged membrane. Prolonged transport negatively affected the sperm quality in terms of both motility and chromatin structure stability. Fertility of sows was affected by semen quality in terms of concentration and sperm motility parameters, as well as ratio of live sperm cells population with damaged acrosome.

Factors influencing the response of spermatozoa to agitation stress: Implications for transport of extended boar semen

The shipping of liquid preserved semen is common practice in animal breeding and prior to cryopreservation for gene banking. Vibration emissions during transport may be harmful to spermatozoa. Therefore, strategies to minimize agitation-induced sperm injury are needed. The aim was to examine whether the type of semen extender, time after semen processing and the temperature in simulated transport conditions influence the response of boar spermatozoa to agitation stress. In Experiment 1, boar semen samples (n = 16) extended in Beltsville Thawing Solution (BTS) or Androstar Plus (APL) medium were filled in 90 mL tubes and shaken for 4 h at 200 rpm either at 22 °C or 17 °C. Samples were then stored at 17 °C for 144 h. In Experiment 2, semen samples (n = 11) extended in Androstar Premium were shaken either directly after filling at 22 °C or 20 h later after cooling to 5 °C. Samples were stored at 5 °C for 144 h. In Experiment 1, sperm motility and viability were lower (p < 0.05) in the shaken samples compared to the controls. The temperature, extender and the storage length had no effect on the agitation-induced loss of sperm quality. Sperm quality traits were higher in samples stored in APL compared to BTS. In Experiment 2, sperm motility at 24 h was reduced (p < 0.05) in those samples shaken at 22 °C but not at 5 °C. Sperm viability, membrane fluidity and mitochondrial membrane potential were not affected in either of the treatment groups. Extended boar semen designed for 17 °C storage and shipped on the day of collection is sensitive to agitation stress. In contrast, spermatozoa slowly cooled to 5 °C and shaken 20 h after processing are more resistant to agitation-induced shear forces and interfacial phenomena.

Effects of air exposure and agitation on quality of stored boar semen samples.

The objective of this study was to investigate the effects of semen volume, air contact inside semen dose tubes, daily agitation of semen doses and extender type on semen quality, thermo-resistance and bacteria growth in extended boar semen doses preserved over 7days of liquid storage. Ejaculates from 4 proven terminal cross-bred boars were collected using the gloved-hand technique for 4weeks and used in the 3×2×2 factorial study. The effects of treatment (CON: 80ml doses sealed at the top of the tube; 40HIGH: 40ml doses sealed at top of tube, and 40LOW: 40ml doses sealed at top of the liquid), agitation (agitated versus not agitated) and extender type (long-term versus short-term) were investigated on semen quality, thermo-resistance and bacteria growth in boar semen doses. The results of the study revealed that motility (p=.031) and viability (p=.041) in 40HIGH were lower than CON. pH (p<.001) was higher in 40HIGH compared with CON and 40LOW. Agitation did not impact motility (p=.581), progressive motility (p=.870), viability (p=.509) or morphology (p=.970), while long-term extender maintained higher motility (p=.002), progressive motility (p=.036), viability (p<.001) and normal acrosome (p<.001) than a short-term extender. VAP (p=.039) of 40HIGH was lower than CON in a thermo-resistance test. Neither treatment (p>.798, .766) nor agitation (p>.396, .476) impacted bacterial growth in this study. In conclusion, air contact negatively impacts boar semen pH and consequently sperm motility. Semen doses prepared with 80 or 40ml volumes of extended boar semen with minimal air contact in the tubes yield more desirable semen quality and agitating boar semen doses daily does not have negative or positive effects on boar semen quality.

PSVI-9 Effects of Mixing Prior to Automated Packaging in Extended Boar Semen

Abstract It is known that sperm cells will settle out of extender over time which could impact the concentration per dose within a batch. The objective of this study to investigate the effects of mixing prior to packaging on concentration of packaged insemination doses. At a commercial boar stud, three ejaculates were collected, extended, pooled and assigned to one of three treatment groups: pour-mix (PM) where ejaculates were immediately packaged after combining the three ejaculates, stir-mix (SM) where three ejaculates were combined and stirred thoroughly then packaged, or no mixing (NM) where pooled ejaculates were allowed to sit for 10 min then packaged. Semen was packaged with 35ml per tube creating ~350 individual doses of semen. Every fifth dose was labeled with consecutive numbers (~70/treatment) and overnighted to Purdue University. Concentration of each dose was determined using the NucleoCounter® SP-100TM. The entire study was repeated over 6 weeks. Within treatment, concentration did not differ among doses for the PM and SM treatments. However, NM doses were less concentrated (P&amp;lt; 0.001) in doses &amp;gt;300 compared to all others and doses 251–300 were less concentrated than doses 1 to 50. NM samples 1–250 averaged 36.12 million cells/ml whereas samples 251 to the end of the batch averaged 34.68 million/ml. This results in the last 20 doses having ~50.4 million less cells/dose. Across treatments, NM had lower concentrations (P=0.02) from doses &amp;gt;200 compared to the SM treatment. PM appeared to be an intermediate as it was numerically lower than SM, but not statistically different from either NM or SM. The difference in concentrations between the NM and SM &amp;gt;200 was 1.85 million/ml equaling 64.75 million fewer cells. Overall, allowing semen to settle and not mixing prior to packaging can result in 50–64 million cells fewer per dose in the last 50–100 doses packaged.

PSV-8 Effects of Air Exposure and Agitation on Quality of Stored Boar Semen Samples

Abstract Recent adoption of post cervical artificial insemination facilitates lowered concentrations of semen inseminated. In order to achieve this reduced concentration, the volume of the insemination dose has been reduced and replaced with additional air in the semen storage container. The objective of this study was to investigate the effects of semen volume, air contact inside semen dose tubes, daily agitation of semen doses and extender type on semen quality, thermo-resistance and bacterial growth in extended boar semen doses over 7 days of liquid storage at 17 C. Ejaculates from 4 terminal cross-bred boars were collected for 4 weeks and used in the 3 x 2 x 2 factorial study. The effects of treatment (CON: 80ml doses sealed at the top of the tube; 40HIGH: 40ml doses sealed at top of tube; and 40LOW: 40ml doses sealed at top of the liquid), extender type (long-term vs short-term), and agitation (agitated vs not agitated) were investigated. The result of the study revealed that motility (P=0.014) and viability (P=0.007) in 40HIGH were lower than CON. pH (P&amp;lt; 0.001) was higher in 40HIGH compared to CON. Agitation did not impact motility (P=0.541), viability (P=0.406) or morphology (P=0.970) while long-term extender maintained higher motility (P=0.034), viability (P&amp;lt; 0.001) and normal acrosomes (P&amp;lt; 0.001) compared to short-term extender. VAP (P=0.039) of 40HIGH was lower than CON in a thermo-resistance test. Bacteria were cultured on both sheep’s blood and MacConkey agar and neither treatment (P=0.798; 0.766) nor agitation (P=0.396; 0.476) impacted bacterial growth in this study. In conclusion, semen doses prepared with 80mL or 40mL of total volume with minimal air contact in the tubes yield more desirable semen quality, as air contact negatively impacts boar semen pH and sperm motility. Additionally, regardless of volume or air exposure, daily agitation of boar semen doses did not affect semen quality.

Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry.

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Low temperature preservation: Influence of putative bioactive microalgae and hop extracts on sperm quality and bacterial load in porcine semen

The frequent use of antibiotics in artificial insemination promotes the development of antibiotic resistance. Therefore, alternatives must be considered. In this study, putative bioactive microalgae and hop extracts were examined to estimate their effect on quality and bacterial load of preserved porcine semen when used a low-temperature storage at 5 °C in Androstar® Premium (ASP) extender (Minitüb). Firstly, five algae extracts (Neochloris oleoabundans (NEO), Pseudococcomyxa simplex (PSEY), Phormidium ambiquum (PHA), Schizochlamydella minutissima (SCH), Cyanidioschyzon merolae (CYA)) and one hop extract (Humulus lupulus (HOP)) in four concentrations (0.2, 2, 20 and 200 μg/mL) were tested on their sperm-compatibility at 17 °C. All extracts in the concentrations 0.2 and 2 μg/mL appeared to be sperm compatible. After 72 h of storage at 5 °C, variants showed an increase of total bacterial subcultures from 103 (ASP w/o supplements) to 113/136/135/136/126/118 (ASP w/NEO/PSEY/PHA/HOP/SCH/CYA). Even though bacterial growth was not inhibited in general, all extracts showed an antimicrobial effect on specific bacteria, including Gram-positive (Trueperella pyogenes, Streptococcus porcinus) and Gram-negative bacterial species (Alcaligenes faecalis, Pseudomonas aeruginosa, Ralstonia sp., Pasteurella sp., Proteus sp., Providencia stuartii, and Escherichia coli). While the proportion of these species was 10% in the control (ASP w/o supplements), their proportion in the supplemented variants ranged from 8% (NEO, HOP) to as low as 3% (PSEY). Although the tested extracts are not suitable to replace conventional antibiotics due to their lack of broad-spectrum antimicrobial activity, they nevertheless present promising opportunities for bacteria-specific, customizable usage in boar semen extenders combined with antibiotics or other antimicrobial agents.

Lysine acetylation participates in boar spermatozoa motility and acrosome status regulation under different glucose conditions

Efficient artificial insemination (AI) with liquid preserved boar semen is essential for the swine industry. Glucose is the most widely utilized energy source in refrigerated boar semen extenders. However, the relationship between glucose concentration and spermatozoa quality is not fully understood. In the present study, boar spermatozoa were used as a model to investigate the impact of different glucose concentrations on spermatozoa motility, mitochondrial activity, and acrosome integrity at physiological temperature (37 °C) and during refrigeration (17 °C). The proportion of progressively motile spermatozoa and mitochondrial activity in the high glucose group were significantly lower than in the low glucose group when incubated at 37 °C for 3 h or 17 °C for 3 d, but not at 17 °C for 7 d. Lysine acetylation is a reversible post-translational modification that plays a crucial role in spermatozoa function. Our results show that spermatozoa protein acetylation levels were higher in the high glucose group than in the low glucose group. The proportions of progressively motile and acrosome-intact spermatozoa were higher in acetyltransferase inhibitor (WM-1119)-treated spermatozoa than in the control. Spermatozoa acetyl-CoA concentration, which is directly linked to acetylation, was significantly higher in the high glucose group than in the low glucose group. Taken together, spermatozoa motility and acrosome integrity can be altered by changing the concentration of glucose in the extender. High glucose concentration-induced lysine acetylation participates in the regulation of boar spermatozoa motility and acrosome integrity during preservation. These results can provide insights into spermatozoa preservation and AI in the swine industry.

Iron Oxide Nanoparticles as an Alternative to Antibiotics Additive on Extended Boar Semen.

This study examined the effect of Fe3O4 nanoparticles on boar semen. Beltsville thawing solution without antibiotics was used to extend ejaculates from 5 boars (4 ejaculates/boar). Semen samples of control group (C) and group with Fe3O4 (Fe; 0.192 mg/mL semen) were incubated under routine boar semen storage temperature (17 °C) for 0.5 h and nanoparticles were removed by a magnetic field. Before and after treatment, aliquots of all groups were cultured using standard microbiological methods. The samples after treatment were stored (17 °C) for 48 h and sperm parameters (computer-assisted sperm analyzer (CASA) variables; morphology; viability; hypo-osmotic swelling test (HOST); DNA integrity) were evaluated at storage times 0, 24, 48 h. Semen data were analyzed by a repeated measures mixed model and microbial data with Student’s t-test for paired samples. Regarding CASA parameters, Fe group did not differ from C at any time point. In group C, total motility after 24 h and progressive motility after 48 h of storage decreased significantly compared to 0 h. In group Fe, linearity (LIN) after 48 h and head abnormalities after 24 h of storage increased significantly compared to 0 h. The microbiological results revealed a significant reduction of the bacterial load in group Fe compared to control at both 24 and 48 h. In conclusion, the use of Fe3O4 nanoparticles during semen processing provided a slight anti-microbiological effect with no adverse effects on sperm characteristics.

Antibiotic-free extended boar semen preserved under low temperature maintains acceptable in-vitro sperm quality and reduces bacterial load

This study aimed to assess the sperm quality and number of colony-forming units (CFU mL−1) in extended boar semen stored at low temperatures with or without antibiotics. Normospermic ejaculates (n = 34) were diluted in split samples with Androstar® Premium with or without antibiotics (ampicillin and apramycin sulfate). The extended semen doses were stored for 120 h under three storage temperatures (5, 10, and 17 °C). Variables were analyzed as repeated measures using the GLIMMIX procedure, in a factorial design. The extended semen doses under low-temperature storage (5 and 10 °C) had total motility above 75% throughout the storage. The interaction antibiotic × temperature was significant for total (P = 0.004) and progressive motility (P = 0.005). In extended boar semen doses with antibiotics, the total and progressive motility increased as the storage temperature increased (80.2%, 84.5%, and 89.1%; 70.5%, 76.0%, and 82.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). In extended semen doses without antibiotics, the total and progressive motility were lower when stored at 5 °C than at 10 °C and 17 °C (81.8%, 85.4% and 86.6% and 71.9%, 76.7%, 78.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). After the thermoresistance test, total and progressive motility of doses with antibiotics were higher at 17 °C than 5 °C (P < 0.05); however, they were not affected (P > 0.05) by storage temperature in extended semen doses without antibiotics. The number of CFU mL−1 was lower in extended semen doses without antibiotics stored at 5 and 10 °C than at 17 °C (P < 0.05); however, in extended semen doses with antibiotics, no effect of storage temperature was observed (P > 0.05). The bacterial load was greater in extended semen without antibiotics than with antibiotics, regardless of the storage temperature (P < 0.05). The acrosome and sperm membrane integrity were not influenced (P > 0.05) by using antibiotics. A higher percentage of normal acrosomes was observed as the storage temperature increased (93.6%, 94.3%, and 96.8% at 5, 10, and 17 °C, respectively; P < 0.0001). The membrane integrity was higher (P < 0.0001) in extended semen doses stored at 17 °C than at 10 or 5 °C. The pH rose throughout the storage in all the treatments, except in extended semen doses stored at 17 °C without antibiotics, in which a decrease in the pH occurred at 120 h (P < 0.05). Although the sperm quality being negatively affected by low temperatures, the storage of extended boar semen doses at 5 °C is possible since the sperm viability in vitro was maintained for up to 5 days, fulfilling the requirements of semen quality to be used in artificial insemination. Nevertheless, the use of extended semen doses without antibiotics requires the optimization of hygiene procedures during semen dose processing.

Boar sperm quality and oxidative status as affected by rosmarinic acid at 17 °C.

Peroxidation damage induces sublethal injury to boar sperm during preservation. Rosmarinic acid (RA) has already been verified to efficiently protect cells from oxidant-induced injury and to produce significant effect on cryopreservation of semen. Through our experiments, we aim at investigating whether RA has a positive effect on the preservation of pig semen at room temperature. The semen collected from sexually mature Large White boars were preserved at 17°C in Beltsville thawing solution (BTS) supplied. The boar sperm were exposed to 0, 25, 50, 75, 100, 125 and 150μM RA in vitro and the sperm functions were examined. The sperm motility, the acrosome and plasma membrane integrity, the catalase activity (CAT), the total antioxidative capacity (T-AOC) activity and the malondialdehyde content (MDA) were examined at 0, 1, 3 and 5days. The BTS diluent containing RA improved the sperm quality during the process of liquid preservation compared with the control treatment. After 5days of liquid preservation, the addition of RA at 100μM produced an optimal effect on the survival time as well as on the maintenance of motility, acrosome and plasma membrane integrity; T-AOC activity; CAT activity; and the MDA content. Besides, our results in the reproductive experiments showed that the addition of RA at 100μM to the BTS diluent increased the pregnancy rate. These results suggest that the proper concentration of RA in boar semen extenders possibly improves the artificial insemination efficiency by reducing the sperm damage and the subsequent dysfunction during liquid preservation in swine production systems.

153 The effects of parity and oxytocin or prostaglandin F2α added to insemination doses on reproductive performance of pigs bred in summer

Summer is the least favourable season for swine reproduction, mainly due to long photoperiods and heat stress. Such conditions negatively impinge on the reproductive system of sows and boars, manifesting in debilitated uterine and gonadal function. This situation is exacerbated by the dilution of ejaculates in semen extender before AI. Consequently, there is a noticeable decline in piglet productivity of summer-breeding gilts and sows. The main goal of this study was to determine the effect of oxytocin (OX) or prostaglandin F2α analogue (PG) added to boar semen extender on the duration of insemination and reproductive performance of pigs bred in July and August. A total of 144 females (80 gilts and second-parity sows (G+SP) and 64 multiparous sows (M)) were divided into three groups (n=48 per group). The OX (11 G+SP and 37M) and PG (20 G+SP and 28M) groups were inseminated twice (at the onset of behavioural oestrus and 22-24h later) using semen supplemented with 20IU of OX or 5mg of PG, respectively; the controls (33 G+SP and 15M) were artificially inseminated with non-supplemented inseminate doses. Pregnancy was detected ultrasonographically on gestational day 42, and the number and viability of piglets were recorded at farrowing. Proportions were analysed using chi-square test (Brandt and Snedecor formula), and other numerical data were analysed using two-way analysis of variance and least significant difference test to determine the differences between individual mean values (SigmaPlot, Systat Software Inc.). The mean duration of first insemination was shorter (P&amp;lt;0.05) in M females (80±22s) compared with G+SP females (191±26s) inseminated with PG-supplemented semen, whereas the second insemination was shorter (P&amp;lt;0.05) in M females than in G+SP females artificially inseminated with OX-supplemented semen (93±15s compared with 192±28s). The mean pregnancy rate was lower (P&amp;lt;0.05) in control G+SP females (26/33; 85%) than in OX G+SP females (11/11; 100%). The farrowing rate was less (P&amp;lt;0.05) in control females (36/48; 75%) than in OX females (44/48; 92%), and it was less (P&amp;lt;0.05) in PG G+SP females (14/20; 70%) compared with PG M females (26/28; 93%). The M females in the OX group had more (P&amp;lt;0.05) stillborn piglets per litter compared with their G+SP counterparts (0.6±0.1 vs. 0.1±0.1). Overall, PG females had more (P&amp;lt;0.05) weak piglets per litter (1.2±0.2) compared with the control (0.5±0.2) and OX (0.6±0.2) groups. The present results reveal the occurrence of both beneficial and undesirable effects of PG and OX added to boar semen extender on reproductive performance of breeding pigs in summer. Addition of PG was associated with shorter first-insemination times in older sows compared with G+SP animals but also with lower farrowing rates in younger animals and an overall increase in the number of weak piglets at farrowing. Supplementation of OX was in turn associated with a shorter second insemination and higher pregnancy rates in young females but more stillborn piglets per litter in multiparous sows. The specific causative mechanisms of these associations remain to be elucidated.

Impact of hygiene on bacterial contamination in extended boar semen: An eight-year retrospective study of 28 European AI centers

Antibiotic agents such as gentamicin represent essential components of semen extenders in order to reduce bacterial contamination. But antibiotic resistance increases and AI centers start utilizing antibiotic agents which are more potent. Therefore, a shift to preventing bacterial contamination has to take place. In this study, we could demonstrate that hygiene is a tool capable of reducing bacterial load. In order to analyze 1434 extended semen samples and nine specially established hygienic critical control points (HCCPs, n = 828), 92 quality control audits have been carried out in a time period from 2012 until 2019 in 28 European AI centers. The results show the process of introducing a basic hygienic standard in audit 1 (2012/2013) and 2 (2014/2015) and the resulting achievements by means of improved hygienic conditions in audit 3 (2016/2017) and 4 (2018/2019). Within the scope of audit 1, 19% of the semen samples were contaminated with bacteria (cutoff ≥100 colony-forming units/mL). Audit 2 showed a bacterial load of 13.6% whereas during audit 3 and 4 very low bacterial contamination rates were recorded (4.5 and 5.5%, respectively). In the same manner, analysis of hygiene at different CCPs during semen production showed a decrease in all average HCCP-scores (score 1–6) comparing audit 4 to 1. By regression analysis we could show a significant audit-dependent association of the bacterial contamination in semen samples and hygiene of HCCPs. Furthermore, analysis of the odds ratio (OR) reveals that the bacterial contamination of certain HCCPs poses an increased risk of receiving bacterially contaminated semen samples (filling machine: OR = 3.02, P = 0.06; extender: OR = 8.97, P < 0.001; inner face of dilution tank lids: OR = 3.14, P = 0.09). Around 60% of the variance of the bacterial contamination in semen samples could be explained by hygienic conditions at different control points and their interaction with audit period and AI center. Antimicrobial agents are essential to protect human and animal health but excessive or inappropriate use can lead to the emergence of resistant bacteria. As shown in our study, hygiene management can significantly reduce bacterial contamination and is therefore capable of preventing antibiotic resistance.

PSII-20 Effect of mixing of extended boar semen using a bulk semen delivery system after 2 and 5 days in storage

Abstract With the increase in utilization of bulk semen delivery systems, the objective of this study was to determine whether differences in semen quality exist from mixing stored bulk semen. Semen with motility and normal morphology &gt;75% were used to create 4, 70-dose and 4, 35-dose bulk semen bags. Semen was cooled and transported 235 miles. On d2 or d5, a bag was mixed and hung for 15 min. Doses were removed every 2 minutes. One 70-dose (70M) and one 35-dose bag (35M) were mixed every 5th dose, while the remaining 70-dose (70NM) and 35-dose (35NM) bags were not mixed. Doses were weighed and analyzed for concentration using CASA and agglutination assessed. Every 5th dose was warmed and evaluated for motility. One milliliter of semen from the 5th dose was preserved and evaluated for sperm cell morphology. Doses averaged 0.04 ± 0.0001 x 109/mL cells in 31.5 ± 0.077 mL resulting in 1.26 billion sperm/dose. All bags averaged &gt;80% motility on d2 and 69% on d5 with &gt;90% normal morphology on both days. There were minor differences in concentration and normal morphology from variation in preparing bags, not associated with mixing. On d2, mixing had no significant effects on agglutination or motility (P &gt; 0.05). Concentration differed between 35M and 35NM (0.0385 ± 0.0004 vs 0.0417 ± 0.0002 x 109/mL; P &lt; 0.001), with no identifiable trend. On d5, mixing had no effects on concentration, normal morphology or agglutination (P &gt; 0.05). Bag 70M had more distal droplets compared with 70NM (3.78 ± 0.31 vs 2.36 ± 0.34%; P = 0.023). There was a non-significant linear decrease in concentration in the 70NM and 35NM bags, where concentration decreased by 0.124 x109 cells. Mixing alleviated this reduction in the 35-dose bags and partially alleviated it in the 70-dose bags. These data demonstrate that mixing had minimal impact on semen quality and was able to mitigate the decrease in concentration in the 35-dose bag.

Supplementation of salvianic acid A to boar semen extender to improve seminal quality and antioxidant capacity.

The purpose of this test was to investigate the effect of salvianic acid A (SAA, CAS No. 76822-21-4) on the quality of boar semen during liquid storage at 17°C. The effects of different concentrations of SAA on semen quality and antioxidant capacity were analyzed. Boar semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 15, 30, 45, 60, 75μM of SAA). During the storage period, sperm activity was measured every 24hr, and plasma membrane integrity, acrosome integrity, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, and catalase (CAT) activity were measured at 0, 1, 3, and 5days. The results from our study suggest that different concentrations of SAA have different effects on semen preservation. Semen samples supplemented with SAA showed reduced effects of oxidative stress on sperm compared to the control samples. Supplementation of 30μM of SAA significantly improved sperm motility, plasma membrane integrity, acrosome integrity, and antioxidant capacity. However, the addition of SAA to the extender was scarcely beneficial to the improvement of results of artificial insemination with boar semen after liquid preservation. Further studies are necessary in order to demonstrate that SAA has good effects on the liquid preservation of semen.

Nanotechnology-based approach for safer enrichment of semen with best spermatozoa

BackgroundAdvances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles (MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic (annexin V) and acrosome-reacted (lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field (nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts (0, 87.5, and 175 μg) of MNP-conjugates (Annexin V-MNP or Lectin-MNP) and incubated (10 to 15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated (0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates (87.5 μg – 30 min) on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.ResultsTransmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved (P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between (P > 0.05).ConclusionsThe findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable, and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.

Storage of boar semen at 16-18 °C in the long-term commercial extender prepared with deionized water or nanowater.

The aim of this study was to assess the usefulness of nanowater (NW; water declusterized using cold plasma treatment) as a diluent for a commercial boar semen extender during the 15-day storage (Days 1 to 15) at 16-18 °C. Ejaculates collected from 8 boars were subjected to the standard evaluation and then diluted in the extender prepared with deionized water (DW) or NW to a final concentration of 3×109 spermatozoa/ml. The proportion of defective spermatozoa increased (P<0.05) from Day 10 to Day 15 of storage (22.8±16.6% to 41.8±26.4% in DW group and 18.6±11.7% to 34.8±25.4% in NW group) and it was significantly greater in DW group compared with NW group on Days 5 and 10 due mainly to a greater (P<0.05) number of mid-piece defects in semen stored in the DW-containing extender. Sperm progressive motility decreased (P<0.05) in both groups between Days 2 and 6, Days 6 and 10, and Days 10 and 12, whereas the percentage of motile spermatozoa declined (P<0.05) to Day 14 only in NW group. Sperm motility was greater (P<0.05) in NW group compared with DW group from Day 5 to Day 13. A decline in sperm progressive motility below 40% in all semen samples occurred by Day 11 in DW group and by Day 12 in NW group. The mean survival time of sperm at 37 °C ex situ was greater in NW group than in DW group on Day 5 (314±87 min compared with 284±87 min) and Day 10 (223±34 min compared with 182±27 min; NW group compared with DW group, respectively). There were no differences (P>0.05) between the two groups in the concentrations of alkaline phosphatase and aspartate aminotransferase in semen extender. To summarize, the use of NW as an extender diluent exerts cytoprotective effects on boar spermatozoa and delays a decline in sperm progressive motility.

Detection and characterization of Lactobacillus spp. in the porcine seminal plasma and their influence on boar semen quality.

The presence of pathogenic bacteria in ejaculates has been a topic in boar semen preservation over the last decades. Since little information is available on commensal bacteria in boar semen, the aim of the present study was to identify commensal lactobacilli in fresh cryopreserved boar semen and to examine their influence on boar semen quality. Therefore, 111 boar ejaculates were investigated for the presence of Lactobacillus species. Thirty samples (27%) contained viable Lactobacillus species (e.g. L. amylovorus, L. animalis, L. reuteri and Weisella minor). L. animalis and L. buchneri DSM 32407 (isolated from the bovine uterus) qualified for further examinations based on their growth rate in six antibiotic-free boar semen extenders. After a 120 min short-term incubation with an antibiotic-free BTS-extender, progressive motility was diminished (P = 0.001) upon addition of 105 and 106 colony forming units (CFU/mL) L. animalis. The supplementation with L. buchneri DSM 32407 had no significant (P > 0.05) influence on sperm quality during short-term co-incubation. After 168 h long-term co-incubation, motility analysis revealed a negative (P = 0.026) impact of 105 CFU/mL L. buchneri DSM 32407. A concentration- and storage-dependent effect is particularly obvious (P < 0.001) using 106 CFU/mL L. buchneri DSM 32407. Most notably, the thermo-resistance (TRT) for 106 CFU/mL L. buchneri DSM 32407 (P = 0.001) was inferior to BTS with and without gentamicin after 72 and 168 h of semen co-incubation. The supplementation of 105 CFU/mL L. buchneri DSM 32407 impaired progressive motility to a lesser extent. The percentage of mitochondrially active spermatozoa after 96 h (P = 0.009) and membrane-intact spermatozoa after 168 h (P < 0.001) was lower when 106 CFU/mL L. buchneri DSM 32407 were suspended compared with all other groups. Finally, the addition of L. buchneri DSM 32407 to BTS-extended boar semen had no competitive effect on the total amount of bacteria 48 h after co-incubation. In summary, the present study demonstrated that there are Lactobacillus species present in the porcine seminal plasma, which can be cultivated using standard procedures. However, long-term co-incubation of lactic acid bacteria with spermatozoa had a negative influence on spermatozoa.

Effect of Astragalus polysaccharide addition to thawed boar sperm on in vitro fertilization and embryo development

It is important to utilize an antioxidant to ameliorate oxidative damage during boar sperm cryopreservation and thawing. Some studies have shown that Astragalus polysaccharide (APS) has antioxidant capabilities in sperm storage at low temperatures. However, the effects of APS on thawed sperm are unclear. In this study, the effect of supplementation of thawing boar semen extender with APS (0.5, 1, 5, 10 mg/mL) on sperm quality parameters (viability, motility, acrosome integrity and mitochondrial activity) was evaluated. Next, we investigated the effect of APS (0.5 mg/mL) supplementation on antioxidant parameters. Semen from two straws was thawed and diluted with three volumes of Beltsville Thawing Solution (BTS) and immediately divided into a control group without addition of antioxidants (CTR) and the APS group. Sperm and antioxidant parameters were evaluated in the CTR and APS groups after 1 h of incubation at 37 °C. Finally, we studied the effect of APS (0.5 mg/mL) supplementation on in vitro fertilization (IVF) and embryo development. The addition of different doses of APS to thawed sperm did not induce any significant effects on the sperm viability or motility compared to the sperm without APS treatment. However, the addition of 0.5 mg/mL APS to thawed sperm showed improved mitochondrial activity, higher penetration rate and increased total IVF efficiency compared with those of the control group. Moreover, our results indicate that the supplementation of APS in thawed sperm decreased the concentration of reactive oxygen species (ROS) and improved the activity of superoxide dismutase (SOD) and catalase (CAT). Finally, the addition of APS significantly increased the cleavage rate and blastocyst rate compared to those of the control group. In conclusion, the addition of APS to thawed boar sperm can enhance the antioxidant ability of sperm and improve in vitro fertilization (IVF) parameters and the outcomes of embryonic development. These results imply that APS has practical potential to enhance boar sperm reproductive performance.

A PCR detection method for discerning Serratia marcescens in extended boar semen

Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.