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 7 days of liquid storage. Ejaculates from 4 proven terminal cross-bred boars were collected using the gloved-hand technique for 4 weeks and used in the 3 × 2 × 2 factorial study. The effects of treatment (CON: 80 ml doses sealed at the top of the tube; 40HIGH: 40 ml doses sealed at top of tube, and 40LOW: 40 ml 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 40 ml 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.     

Bacterial killing activity and lysozymes: A stable defence mechanism in stallion seminal plasma?

During insemination, bacterial contamination of the ejaculate can lead to reduced sperm quality and transmission of pathogens to the female, thus should be avoided. The semen of a variety of animal taxa possess antimicrobial properties against a wide range of bacterial species through antimicrobial molecules, such as lysozyme, but their variance and the factors influencing it are unknown for most species. In this study, the antibacterial defence (bacterial killing activity (BKA) against Escherichia (E.) coli and Staphylococcus (S.) aureus as well as lysozyme concentration) was studied in seminal fluid from two consecutive ejaculates of 18 stallions. All ejaculates showed BKA against the tested bacteria, which correlated between the two consecutive ejaculates (r_S = 0.526, p = .025 for E. coli and r_S = 0.656, p = .003 for S. aureus) and appeared to be stable over the tested period. The lysozyme concentration (LC) showed no significant correlation between the consecutive ejaculates (r_S = 0.161, p = .681). However, LC had a positive correlation to the ratio of apoptotic spermatozoa within the ejaculates (r_S = 0.426, p = .019). In contrast to other livestock (e.g., boar, bull), the BKA in stallion semen did not correlate significantly with the age of the animal nor sperm quality characteristics.     

Effect of pasteurized egg yolk on the quality of cryopreserved boar semen

Egg yolk (EY, control) is an essential ingredient of diluents for boar semen cryopreservation. Pasteurized egg yolk (PEY) reduces hygienic risks in processing and is easier to standardize. The aim of this study was to evaluate the in vitro effect of PEY (treatment) on frozen-thawed boar semen. In a split-sample approach (n = 13 boars), it could be shown that there is neither an influence (p > .05) on post-thawing motility (PTM: 5, 30 and 120 min) nor on morphologically intact sperm, percentage of acrosome defects and membrane fluidity using a PEY extender compared to the control. Mitochondrial activity (p = .043), membrane integrity (p = .015) and PTM 300 min (p = .023) were slightly affected in the treatment group. Overall, sperm quality was at a high level in both experimental groups. Further studies are needed to determine the impact of PEY on the fertilizing capacity of boar ejaculates.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.     

Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen

Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC without antibiotics (CW and SW, respectively). Bacteriological examinations were carried out within 2 hr. Thirty‐one species of bacteria were isolated from one or more ejaculates, with Corynebacterium spp. being the most frequently detected. Corynebacterium spp. were present in all ejaculates. The MSLC resulted in a significantly lower total bacterial count than controls (CA vs. SA, p < 0.001; CW vs. SW, p < 0.0001).     

Single Layer Centrifugation with 20% or 30% Porcicoll separates the majority of spermatozoa from a sample without adversely affecting sperm quality

Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16–18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p > .05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p > .05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p < .05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p < .05- <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p < .05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling-up to process larger volumes of boar ejaculates.     

Irradiation of semen doses with LED‐based red light in a photo chamber does not improve in vitro quality of thermically stressed boar spermatozoa

Recent reports indicate that stimulation of liquid‐stored boar semen with red LED‐based light improves sperm quality and reproductive performance in sow herds. So far, in vitro data after LED stimulation of whole semen doses are lacking. In this study, the effect of LED light exposure on the in vitro quality of boar spermatozoa after storage and thermic incubation was examined. Boar semen doses were stored at 17°C (n = 10) or 5°C (n = 6) in Beltsville Thawing Solution extender and then exposed to red LED light using a commercial photo chamber. During a subsequent long‐term incubation at 38°C, neither sperm kinematic parameters nor mitochondria function or membrane integrity differed between control and treated samples (p > .05). It is concluded that stimulation of semen doses in the LED‐photo chamber does not improve quality of thermically stressed boar sperm in vitro. Other than the sperm traits tested here might be involved in the previously reported improvement of in vivo fertility.     

Linseed oil in boar's diet improved in vivo fertility and antioxidant status

The reactive oxygen species (ROS) which are produced during storage of boar semen are causing oxidative stress and leads to poor fertility. Also, tropical and sub-tropical weather condition adversely impacts the physicomorphological quality and fertility of boar sperm. The aim of this study was to examine the effects of feeding linseed oil to boar on its seminal attributes, sperm kinetics, biomarkers of antioxidant, fatty acid profile of seminal plasma (SP) and sperm and in vivo fertility. Six Hampshire crossbreed boars were fed with 90 ml linseed oil (LIN) whereas six Hampshire crossbreed boars were fed 90 ml canola oil (CON) for 16 weeks. Sperm quality was evaluated (60 ejaculates for each group; a total of 120 ejaculates) for motility, livability, abnormal morphology, acrosomal membrane integrity, hypo-osmotic swelling test (HOST) and sperm kinetic parameters by computer assisted semen analysis (CASA) at 0 h and at 72 h of storage at 17°C. Biomarkers of antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC) and malondialdehyde (MDA) were measured in SP and serum. Gas chromatography–mass spectrometry (GC–MS) was used for the estimation of fatty acid composition of SP and sperm. Boars fed with linseed oil had higher semen volume (p < .01) and more total sperm numbers (p < .01). Feeding linseed oil to boar enhanced seminal attributes (p < .05) at 0 h as well as at 72 h of storage. Linseed oil feeding (p < .01) improved biomarkers of antioxidants and significantly (p < .01) lowered the lipid peroxidation in serum and SP. Linseed oil feeding (p < .05) increased the proportion of alpha linolenic (ALA), arachidonic and docosahexaenoic (DHA) fatty acids in SP. The ratio of n-6 to n-3 fatty acids in sperm increased significantly (p < .01) in treatment group. Farrowing rate was significantly (p < .05) higher in treatment group. In conclusion, feeding linseed oil to boar improved the in vivo fertility, enhanced antioxidant capacity and increased the DHA content of SP and sperm.     

Dynamics of sperm DNA fragmentation in raw boar semen and fertility

The aims were to evaluate sperm DNA fragmentation (SDF) in boars through the dispersion of their chromatin in raw semen samples, quantifying the extent of SDF, and to assess dynamic aspects of sperm DNA damage after incubation to obtain the rate of sperm DNA fragmentation (rSDF) under thermal conditions similar to the uterus (37°C) over a period of up to 24 hr and to correlate the reproductive outcome of the sows with the SDF of the boars at ejaculation. The study was performed on a pig‐breeding farm in southern Uruguay. Sixty‐one ejaculates from five of the most frequently used hybrid boars were evaluated. Semen was collected weekly from each of the boars, using the gloved‐hand technique and discarding the jelly‐like fraction of the ejaculate. Fresh semen was kept in a water bath at 37°C and protected from light, and was thereafter processed with Sperm‐Sus‐Halomax^® to evaluate SDF. The smears for time 0 (T0) were made on farm, and thereafter smears were made at the laboratory at 4 hr of obtaining the semen (T4), then every 2 hr (T6, T8, T10, T12) and a final fixation at 24 hr (T24). Differences in SDF were observed among exposure times for all boars (p < .05), but not between T10 and T12 (p = .7751) nor T4 and T24 (p = .9113). In none of the T24 samples, sperm heads could be seen with chromatin dispersion halos. Furthermore, there were differences among boars when comparing sperm rSDF (p < .05). Farrowing rate was not affected by SDF at T0 (r = .38, p = .75), nor was litter size (r = .16, p = .70). With the present experimental conditions, we have not been able to show a relationship between sperm DNA fragmentation at ejaculation and reproductive performance. However, this could be a result of the low number of ejaculates and boars used.     

Effects of sulfanilamide on boar sperm quality,bacterial composition,and fertility during liquid storage at 17°C

Sulfanilamide (SA) is an effective broad‐spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum)

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step‐down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.     

Antibiotic resistance in microorganisms isolated in a bull semen stud

Many microorganisms from various sources may be present in ejaculates of bulls. This study identified and isolated bacteria from bull sperm samples in a commercial stud and evaluated their resistance to antibiotics. The number of colony-forming units was determined in semen samples collected at distinct steps during freezing and thawing. The minimum inhibitory concentration and the minimum bactericidal concentration were determined for four antibiotics commonly used in commercial studs. A total of 135 microorganisms from 25 genera were isolated. After a sensitivity test, all evaluated microorganisms (n = 55) were resistant to penicillin and most of them were resistant to tylosin and lincomycin (n = 54). Resistance to all tested antibiotics was observed in 22% of all isolates, whereas only 3.9% of the isolates were inhibited by the tested antibiotics at the concentrations recommended by the international legislation. As the isolated microorganisms presented high resistance to frequently used antibiotics, sensitivity tests should be periodically conducted in commercial bull semen studs to prevent the use of contaminated semen in artificial insemination.     

Assessment of Risk Factors in Milk Contamination with Staphylococcus aureus in Urban and Peri‐Urban Small‐Holder Dairy Farming in Central Ethiopia

Assessment of risk factors associated with milk production systems is central to ensuring quality and safety of milk and milk products. This study was aimed at identifying possible risk factors in milk contamination in urban and peri‐urban areas of the central high lands of Ethiopia. A total of 477 on‐farm pooled milk (n = 433) and combined bulk milk samples (n = 44) were collected and processed using standard microbiological techniques to isolate and characterize Staphylococcus aureus. In addition, 433 individual farm owners and 22 collection centre owners were interviewed using a structured and pre‐tested questionnaire. Multivariate logistic regression was used to determine risk factors. Of the total individual on‐farm pooled milk samples analysed (n = 433), it was found that 103 of the individual milk samples (24%) and 17 of the combined bulk milk (39%) were positive for S. aureus. This difference in prevalence was statistically significant. Even though there were a number of potential variables associated with the recovery of S. aureus in bovine milk, four variables including cleaning milk container with hot water and detergent [Adjusted OR: 0.342, 95% CI, (0.166, 0.701)], mastitis check [Adjusted OR: 3.019, 95% CI (1.542, 5.913)], travel time to collection centres [Adjusted OR: 4.932, 95% CI, (2.265, 10.739)] and amount of milk delivered by farmers to collection centres per day [Adjusted OR: 1.059 (1.032, 1.087 β = 0.057)] were found to be statistically significantly associated with isolation of S. aureus. We recommend a targeted educational intervention on defined risk factors to reduce the post‐harvest S. aureus contamination of raw milk in urban and peri‐urban milk shed areas of central Ethiopia.     

The Combinatorial Effect of Different Equex STM Paste Concentrations,Cryoprotectants and the Straw‐Freezing Methods on the Post‐Thaw Boar Semen Quality

This study was to evaluate the combinatorial effect (14 treatments, A–N) of different Equex STM paste concentrations, cryoprotectants and the straw‐freezing method on the post‐thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post‐thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post‐thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw‐freezing method changes between glycerol and DMA.     

Multivariate Analyses for Determining the Association of Field Porcine Fertility With Sperm Motion Traits Analysed by Computer‐Assisted Semen Analysis and With Sperm Morphology

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate – FR and total piglets born – TB) of 14 boars was obtained from a field trial conducted during 10 week of breeding period on a commercial farm using multiparous sows (n = 948) through single‐sire mating with 2 × 10⁹ motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer‐assisted semen analysis system in raw and stored semen at 17°C for 240 h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.     

Is neutral genetic diversity related to quantitative variation in semen traits in bulls?

Conservation decisions based on neutral genetic diversity have been observed to promote retention of useful quantitative variation in biological populations. An experiment was undertaken to determine the association between microsatellite marker polymorphisms and phenotypic variation in semen production and cryosurvival traits in bulls. Thirty-five ejaculates were collected from ten bulls of two breeds and evaluated before and after cryopreservation for several semen traits. The bulls were also genotyped using a set of sixteen bovine-specific microsatellite marker loci. Fixation indices (F_ST), heterozygosity and Nei's genetic distance measures were computed from allele frequency data for each of the bulls. Molecular and phenotypic data were used to compute tri-distance matrices for the ten bulls and correlated using Mantel's test in GenAIEx 6.5. The study revealed extensive heterogeneity in semen traits, heterozygosity and F_ST values among the bulls. Large pairwise phenotypic and genetic distances were also observed. Correlation between pairwise genetic distances and phenotypic distances was significant and highly positive for sperm viability (r = .61, p < .001) and moderately positive for sperm motility (r = .40–42, p < .05) variables. For sperm morphology, ejaculate volume and sperm concentration, correlation with genetic distances was positive, low and not significantly different from zero (p > .05). A tendency for a triangular-shaped relationship between genetic and phenotypic distances for post-thaw motility and viability traits was also observed. Accordingly, association with neutral genetic diversity was absent for semen production traits and moderate to highly positive for sperm cryosurvival traits. Given these findings, conservation decisions based on neutral genetic diversity may capture variation in some adaptive traits, but not others.     

Sanitary Procedures for the Production of Extended Semen

Semen is collected and processed from a variety of animal species for use in artificial insemination breeding programmes. Because of the inherent nature of the semen collection process, bacterial contamination of the ejaculate is a common occurrence. Additionally, manipulation of the ejaculate during processing in the laboratory can expose the sample to possible introduction of bacterial contamination. If preventative measures at the stud fail to adequately control these risks, decreases in semen quality, dose longevity and fertility may occur. Multiple mammalian and non-mammalian sources have been identified as origins of contamination in the stud. Knowledge of these sources has aided the industries in developing strategies that help in controlling the introduction of contaminant bacteria in extended semen. A primary step in minimizing contamination is in the practice of good hygiene by stud personnel. Prudent general sanitation protocols should also be followed in the laboratory, animal housing and semen collection areas. Cleanliness and attention to the actual semen collection process can also aid in reducing bacterial load originating from the stud semen donor. Attentiveness to all of these steps significantly contributes to an overall reduction in the type and amount of bacterial contamination. However, their complete elimination stills remains unavoidable. To address residual bacteria load in the sample, antimicrobials are commonly used in semen extenders intended to promote in vitro sperm longevity beyond that of a few hours. Current research by the animal industries continues in the selection and prudent use of antimicrobials that will lead to the success and sustainability of this modality in controlling bacterial contamination.     

Dose rates of antimicrobial substances in boar semen preservation—time to establish new protocols

To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37 mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5 mg/L, which is close to or below the epidemiological cut‐off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512 mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic‐free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready‐to‐use extenders. In conclusion, an end volume‐based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.     

Effect of semen extenders and storage time on quality of Muscovy duck (Cairina moschata) drake semen during the entire reproductive season

Avian semen dilution with appropriate extender allows to prolong the fertilizing ability of sperm stored in vitro. In the present study, the impact of extenders and time of storage on morphology of Muscovy duck (Cairina moschata) drake semen were examined. Semen was collected twice a week, using male stimulation by a female method, from 12 adults (29 weeks old) drakes kept individually in cages, under controlled environmental conditions. Freshly collected, pooled ejaculates were divided into three part: neat undiluted sample, and diluted 1:1 with Schramm (SCH) or Watanabe (W) extender and stored at 4°C. Morphological examination of all samples was conducted after dilution and then, after 3 and 6 hr of storage. The storage of undiluted semen caused decrease (p ≤ .01) in live morphologically normal sperm, from 79.73% in the freshly collected ejaculates to 55.75% and to 12.12% after 3 and 6 hr of storage, respectively (average calculated for the entire reproductive season). In the semen diluted with Schramm's extender the adequate values attained 86.84, 79.65 and 61.66%, and using Watanabe extender 84.77, 83.58 and 75.25%, respectively. The period of semen storage and the type of extender caused significant (p ≤ 0,05; p ≤ 0,01) changes in sperm morphology. The longer period of storage contributed to the decrease in number of morphologically normal sperm, whereas their content in Watanabe extender after 3 and 6 hr of storage was higher (p ≤ .01) than in semen diluted in Schramm extender.