BackgroundDystocia is an uncommon but potentially life-threatening complication of equine parturition. Due to early separation of the chorioallantois during the expulsive stage, fetal oxygen supply rapidly becomes compromised, making dystocia a time-critical emergency for both mare and foal. Objectives: To describe obstetrical interventions performed at equine referral hospitals, evaluate mare and foal survival, and identify factors associated with survival outcomes in a Swedish population where geographic distances may influence access to specialized veterinary care.MethodsMedical records from three equine referral hospitals in central Sweden were reviewed for dystocia cases presented between January 2006 and December 2022. Data collected included dystocia causes, obstetrical interventions, dystocia duration, prior veterinary intervention, and survival outcomes for mares and foals.ResultsA total of 138 dystocia cases were included. Fetal causes of dystocia predominated (114/138), with malposition being most common (106/138). Controlled vaginal delivery was the most frequently performed intervention (33.3%, 46/138). Of 140 foals delivered, 17.1% were born alive and 12.1% survived hospital discharge. Foal survival was not different among delivery methods (p = 0.145). However, survival was significantly associated with shorter dystocia duration (p < 0.01) and was higher when no obstetrical intervention had been attempted prior to referral (p = 0.0436). Among mares, 23.2% did not survive to hospital discharge, most commonly due to endotoxemia or sepsis.ConclusionDuration of dystocia prior to hospital admission was a key factor associated with neonatal survival, highlighting the importance of early recognition of dystocia, close monitoring of mares approaching foaling, and prompt referral to specialized equine hospitals.

Freezability of stallion spermatozoa varies considerably between individuals. The aim was to investigate whether adding seminal plasma (SP) from good freezer or bad freezer stallions improved post-thaw sperm acrosomal integrity. Semen samples were available where SP-free spermatozoa from good freezer and bad freezer stallions prepared by SingleLayer Centrifugation (S) were treated with pooled SP from the same stallions prior to freezing. Control samples were frozen conventionally (C). Post-thaw acrosomal integrity was assessed by flow cytometry with/without stimulation by calcium ionophore. Higher proportions of live acrosome-intact spermatozoa (p = 0.04) and lower proportions of dead acrosome-intact spermatozoa (p = 0.48) occurred in non-stimulated S samples than in C. Acrosome integrity was not affected by either type of SP. However, in ionophore-stimulated samples, SP from good freezer stallions produced higher proportions of live acrosome-intact spermatozoa than C (p = 0.047), whereas SP from bad freezer stallions resulted in higher proportions of live acrosome-reacted spermatozoa than C (p = 0.04); the proportion of dead acrosome-intact spermatozoa was higher in C than in S (p = 0.05). Thus, S had a lower propensity to acrosome-react under stimulating conditions than C; the effect of adding SP depends on its origin. Further research is required to optimise cryopreservation protocols for stallion semen.

Introduction:Evaluating the vaginal bacterial isolates of dairy cows and their antimicrobial resistance patterns can improve animal health management practices and reduce the risk of transmitting antimicrobial-resistant pathogens within animal populations and from animals to humans. Materials and methods:This study characterized the culturable bacterial composition and antimicrobial resistance (AMR) profiles of vaginal bacteria isolated from healthy dairy cows at the Swedish Livestock Research Centre, Lövsta, Sweden. Unsupervised machine learning techniques, including Partitioning Around Medoids clustering and co-occurrence network analysis, investigated bacterial co-occurrences in the vaginal microbiota. Bacterial isolates were classified as resistant or wild type based on the epidemiological breakpoint values for inhibition zone diameters in the disk diffusion method defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Results:A total of 127 bacterial isolates representing 34 different species were isolated from the samples collected from the anterior vagina of 40 cows. Histophilus somni, Streptococcus pluranimalium, and Escherichia coli were the most prevalent species. The network analysis identified distinct microbial communities, revealing that S. pluranimalium and H. somni were central to microbial interactions within the vaginal microbiota. The disk diffusion susceptibility test identified Staphylococcus spp. isolates (n = 10) as resistant to penicillin (70%), tigecycline (50%), tetracycline (30%), and levofloxacin (30%), and were wild type for ciprofloxacin and trimethoprim-sulfamethoxazole. Streptococcus spp. isolates (n = 18) showed resistance to tetracycline (61.11%), tigecycline (27.78%), penicillin (22.22%), levofloxacin (11.11%), and trimethoprim-sulfamethoxazole (11.11%). Escherichia coli isolates (n = 11) exhibited resistance to ciprofloxacin (90.91%), tigecycline (81.82%), and levofloxacin (18.18%), and were wild type to trimethoprim-sulfamethoxazole. Conclusion:Our study highlights the importance of including commensal reproductive tract bacteria in AMR monitoring programs, as these species may serve as reservoirs of resistance genes and contribute to the spread of AMR within dairy herds.

Cryopreservation of spermatozoa requires the use of cryoprotective agents (CPAs) to minimize membrane damage and preserve cell function after thawing. In South American camelids, glycerol (GL), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) are the most widely used CPAs. However, some studies in other species have explored the potential of amide-based CPAs, such as dimethylacetamide (DMA), dimethylformamide (DMF), and methylformamide (MF). This study aimed to evaluate the interaction between CPA type and concentration on post-thaw sperm quality in alpacas using a 6 × 3 factorial design (six CPAs × three concentrations: 1 %, 3.5 %, and 7 %). Post-thaw assessments included total motility (bright-field microscopy), viability (SYBR14/PI), and mitochondrial membrane potential (MMP) (MitoTracker Deep Red), with the latter two measured by imaging flow cytometry. Data were analyzed using two-way ANOVA to determine main effects and interactions. CPA concentrations of 1 % and 3.5 % produced significantly higher values for motility, viability, and MMP compared to 7 % (P < 0.05). DMSO and GL exhibited significantly higher post-thaw motility (P < 0.05) than DMA and DMF, although no significant differences among CPAs were observed for viability or MMP. In conclusion, CPA concentration has a greater impact than CPA type on post-thaw sperm quality, and concentrations between 1 % and 3.5 % are optimal for preserving motility, viability, and mitochondrial function in alpaca epididymal spermatozoa.

Elevated ammonia and urea, common byproducts of nitrogen metabolism, are increasingly found in dairy cows and may negatively impact reproductive function. However, their effects on bull sperm remain unclear. This study aimed to assess the impact of ammonia and urea at physiologically relevant concentrations on key sperm characteristics. Thawed bull semen was incubated under five treatment conditions: control (no added ammonia or urea), low urea (LU), high urea (HU), low ammonia (LA), and high ammonia (HA). Sperm motility and kinematics were assessed using Computer-Assisted Semen Analysis (CASA), and sperm viability, DNA integrity, mitochondrial membrane potential (MMP), and oxidative stress markers were evaluated using flow cytometry. HA significantly reduced MMP (p = 0.008) and several motility parameters, including progressive motility and velocity (VCL, VSL, ALH), compared to LA and control groups. A decrease in sperm viability was observed in the HA group compared to LA at the beginning of incubation. While HU reduced MMP (p = 0.002), sperm motility was not significantly affected compared to LU. No significant differences were found in DNA fragmentation or oxidative stress biomarkers between groups. These results highlight the impact of ammonia, specifically, on sperm mitochondrial function and motility, which are crucial for successful fertilization.

Although freezing methods have been optimized for preserving sperm integrity, their effectiveness in sustaining bacterial viability is unknown. Therefore, culturing thawed semen samples might not give an accurate picture of the bacteria in the original sample. The aim of this study was to assess how cryopreservation and storage duration influence bacterial populations and the survival of distinct bacterial species. Semen samples were collected from 14 bulls, samples were diluted in equal proportions of antibiotic-free semen extender and transported to the laboratory at 6 °C overnight. Aliquots of semen were cultured within 24 h after semen collection on Plate Count Agar to calculate number of bacteria, and blood agar plates (5 % bovine blood) for identification of bacterial species. The remaining samples were diluted 1:1 in Brain Heart Infusion (BHI) broth with 30 % glycerol and stored at -80 °C. The frozen samples were thawed and cultured for quantification of bacteria as described for fresh semen, after 6 and 13 days at -80 °C. The isolated bacteria were re-cultured on blood agar, incubated for one day at 37 °C before identification by Matrix assisted laser desorption ionization-time of flight mass spectrometry. Total bacterial counts remained consistent across fresh and cryopreserved samples regardless of storage duration. A total of 31 bacterial species were identified, with 20 detected in fresh samples, 16 present after 6 days of storage, and 18 observed after 13 days. Ten species persisted across all time points, while others were unique to a specific sampling day, including nine species on day 1, two species on day 6, and five species on day 13. These findings suggest that while cryopreservation does not alter the overall bacterial load, the survival of individual species varies depending on storage conditions.

Cryopreservation is known to destabilise spermatozoa and is associated with deficiencies in protamine levels and increased DNA fragmentation, which can reduce fertility in various species. The objective of this study was to evaluate the impact of cryopreservation on protamine levels and DNA fragmentation in alpaca spermatozoa. A total of 108 testicles/epididymides were collected from a slaughterhouse and sperm were recovered from the cauda epididymis. Only samples meeting the criteria of > 10 g in weight, > 3 cm in length, > 30% motility, and > 50 million spermatozoa/mL were processed. Sixty samples (n = 60) were suitable for cryopreservation: 30 were used to assess protamine levels, and 30 to evaluate DNA fragmentation. Assessments were conducted both before and after cryopreservation using imaging flow cytometry. Protamine levels were assessed with chromomycin A3 (CMA3, 0.25 mg/mL), where fluorescence inversely correlates with protamination levels. The TUNEL assay was used to analyse DNA fragmentation, following fixation with 0.4% formaldehyde and permeabilisation with 0.8% Triton X-100. Results showed a significant decrease in CMA3 mean fluorescence after cryopreservation (288.19 ± 145.53 mFL vs. 68.54 ± 51.25 mFL, p < 0.05) and an increase in DNA fragmentation (2.98 ± 2.39 vs. 9.45 ± 15.43, p < 0.05). In conclusion, cryopreservation decreases CMA3 fluorescence, related to a possible increase in protamination, and increases DNA fragmentation in alpaca spermatozoa.

Background/Objectives: Alternatives to antibiotics in semen extenders used for pig breeding are needed. Cold storage of boar semen is possible in certain extenders; however, bacteria can still survive. Colloid centrifugation can remove bacteria; therefore, a combination of colloid centrifugation and cold storage on the bacterial load and sperm quality of boar semen was investigated. Results: The sperm quality in the original samples were similar in samples with and without antibiotics, respectively. At Day 4, the bacterial count was not different between controls stored at 17 °C with antibiotics and SLC cold-stored samples without antibiotics. The proportion of dead sperm increased in controls at 16–18 °C (28.12 ± 21.72% vs. 32.88 ± 20.94%; p < 0.05), and DNA fragmentation increased from 1.5 ± 1.02% at Day 0 to 6.1 ± 3.64% at Day 4 (p < 0.01), whereas it did not deteriorate in the cold-stored samples (Day 4: %DFI 2.00 ± 1.65% and 1.90 ± 1.05% for SLC samples with and without antibiotics, respectively). Methods: Aliquots of boar ejaculates were extended in Androstar Premium with and without antibiotics. The samples without antibiotics were processed by Single Layer Centrifugation (SLC) through Porcicoll; sperm pellets were resuspended in AndroStar Premium, with or without antibiotics, cooled slowly, and stored for 4 days at 4 °C. Controls (aliquots extended in AndroStar Premium with antibiotics) were stored at 17 °C. The bacterial load and sperm quality were assessed in the original samples and after 4 days of storage. Conclusions: Single Layer Centrifugation combined with cold storage is an effective method for reducing the bacterial load in boar semen, with or without antibiotics.

IntroductionBacterial contamination can occur at multiple stages of semen processing, necessitating the use of antibiotics in bull semen preservation, mandated by regulatory guidelines. To manage antimicrobial resistance (AMR), targeted antibiotic use based on bacterial identification is essential. This study aimed to characterize bacterial communities in bull semen using metagenomic analysis and MALDI-TOF MS across different semen collection times from the same bulls and between two breeds.MethodsSemen samples were collected from 20 dairy bulls (8 Viking Holstein and 12 Viking Red). Each bull provided three ejaculates within a week: the first after a 96 h since previous collection (T1), the second 48 h later (T2), and the third 24 h after the second (T3). Bacterial species were identified through culturing on cattle blood agar, followed by MALDI-TOF MS identification. Additionally, 16S rRNA sequencing was performed to determine bacterial diversity after DNA extraction.ResultsMALDI-TOF analysis identified 33 bacterial species across 60 semen samples. Six species were exclusive to Viking Holstein (VH) bulls, while 12 were specific to Viking Red (VR) bulls. Certain bacterial species were present only at specific time points: three in the first ejaculate, seven in the second, and five in the third. Across individual bulls, Bacillus spp., Proteus spp., and Staphylococcus spp. were the most consistently detected. Metagenomic analysis revealed 23 phyla and 402 genera in semen samples. Alpha diversity (Shannon index) showed a trend toward p = 0.07 across the bull samples, while beta diversity significantly differed between breeds, with VH samples forming a distinct cluster and VR samples displaying greater microbiome variability. Additionally, specific genera appeared only at one collection time point: Bacteroides, Serratia, Pantoea at T1, Wolbachia, Prevotella, Peptococcus, Alloprevotella at T2, and Streptococcus, Staphylococcus, and Mycoplasma at T3. Specific genera, Acidocella and Escherichia, exhibited negative correlations with most bacterial taxa but showed a slight positive correlation with each other; while Acidocella was detected in nearly all semen samples, except for two samples.DiscussionThe seminal microbiota of bulls varies over time and differs between breeds, indicating that it is influenced by a complex interaction of environmental, physiological, and host-related factors.

Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× g for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation) were evaluated before and after freezing using kinematic and flow cytometric analysis. The parameters for fresh samples were within the normal range for stallion semen but were lower after thawing. There were few differences between the three preparation methods. Interestingly, DNA fragmentation was affected most by the sperm preparation method, being lowest for SLC through high density Equicoll, although SLC through low density Equicoll was effective for some stallions. Some differences were observed in the proportions of live or dead spermatozoa positive for hydrogen peroxide. In conclusion, all of these methods would be suitable for the preparation of semen prior to cryopreservation, but Single Layer Centrifugation through high density Equicoll was the most effective in removing spermatozoa with damaged DNA.

Stringent efforts are being made to restrict the development of antimicrobial resistance by good antibiotic stewardship in medical, veterinary and environmental health. In the pig industry, considerable volumes of antibiotic-containing extender are utilised in artificial insemination. Separating spermatozoa from bacteria by colloid centrifugation followed by cold storage could provide an alternative to antibiotics in semen extenders, provided that sperm fertility is not affected by this combination of techniques. The objective of this proof-of-concept study was to compare reproductive outcomes in sows inseminated with control sperm samples or with samples prepared by Single Layer Centrifugation (SLC) through a low density colloid and cooled to 4 °C. A further aim was to compare the SLC sperm preparations with and without antibiotics. Sows were divided into three groups for insemination as follows: control semen prepared and stored conventionally (n = 16), SLC samples prepared with antibiotics and cold stored (n = 18), SLC samples prepared without antibiotics and cold stored (n = 18). The yield of spermatozoa was 84–93%, respectively. Farrowing rates were 88% for controls and 89% for SLC samples. Mean litter size was 16.6, 17.5 and 17 for controls, SLC with antibiotics and SLC without antibiotics, respectively. Stillborn piglets per litter were also similar between groups (0.7, 1.4 and 1.4 respectively). In conclusion, the combination of techniques did not adversely affect sperm fertility or reproductive outcomes of inseminated sows, regardless of whether antibiotics were included in the resuspended sperm pellets after SLC.

Cryopreservation of sperm is a crucial tool for the long-term preservation of male genetic material, causing significant issues in motility, membrane, and acrosome integrity, among other parameters. Antioxidants have been used to cope with these detrimental effects. We tested 1. the toxicity of a wide range of water soluble beta-carotene (beta: 250-4000 mu M) and alpha-tocopherol (alpha: 31-496 mu M) concentrations on boar ejaculated sperm (n = 6) in parameters as motility, viability, acrosome reaction, apoptosis, oxidation, mitochondrial activation and membrane potential; 2. the effect of various beta-carotene (250-1000 mu M) and alpha-tocopherol (31-124 mu M) concentrations added to the cooling-freezing or thawing extenders (n = 30) before (0 min) and after 90 min incubation (37 C-degrees). Toxicity results showed a decrease in the proportion of live spermatozoa with non- reacted acrosome from 75.1 +/- 3.3 % using beta 250/alpha 31 to 60.1 +/- 5.7 % and 59.3 +/- 5.4 % in samples with beta 2000/alpha 248 and beta 4000/alpha 496 respectively (p < 0.05), suggesting a detrimental effect of the highest concentrations. Antioxidant supplementation in the cooling-freezing extender decreased the apoptotic and oxidized spermatozoa in beta 500/alpha 62 and beta 1000/alpha 124, relative to the control. In contrast, antioxidants addition to the thawing extender induced some detrimental effects in several sperm parameters analyzed. In conclusion, water-soluble beta-carotene and alpha-tocopherol prevent acrosome reaction and oxidation during cooling-freezing on boar sperm. High concentrations of these antioxidants negatively impacted motility and mitochondrial function, suggesting cytotoxic effects and potential capacitation-like changes. The beta 1000/alpha 124 showed protective effects during cryopreservation, but post-thawing supplementation may stimulate oxidative stress rather than prevent it.

IntroductionBacterial contamination of ejaculates during semen collection is practically inevitable, and antibiotics are a constituent of semen extenders. However, bacterial resistance to antibiotics is a serious problem. The present study investigated the effect of preparing semen by centrifugation using a low-density colloid during the preparation of semen doses under field conditions, as an alternative to the use of antibiotics.MethodsFive ejaculates from four boars were each divided into two aliquots: control semen doses, which were extended with a commercial Beltsville Thawing Solution (BTS) containing antibiotics (30 × 106 spermatozoa/mL), and treated semen doses, which were processed with Porcicoll (300 × g; 20 min); the resulting sperm pellet was re-suspended in Beltsville Thawing Solution without antibiotics (30 × 106 spermatozoa/mL). Sperm motility and kinematic variables were assessed for the control and treated semen doses using computer-assisted sperm analysis (CASA). Sows were divided into two groups and inseminated with either the control or treated semen doses. In the second round, after weaning the litters from the first round, the sows in the control group were inseminated with the treated semen doses and those in the treated group received the control semen doses. For all groups, the pregnancy rate, farrowing rate, litter size, number of live-born piglets, and number of weaned piglets were recorded.ResultsNo differences (p > 0.05) between the control and treated semen doses or between the rounds were observed for any CASA-assessed motility and kinematic variables. Pregnancy (p = 0.0271) and farrowing (p = 0.046) rates were higher in the sows in the control group compared to the treated group. No differences were observed in litter size, number of live-born piglets, and number of weaned piglets (p > 0.05) between the control and treated groups, and farrowing rates were the same for the sows in both rounds. Under the current experimental conditions, sperm quality was not impaired by treatment with Porcicoll. An interesting finding is that the sows were able to become pregnant again after insemination with semen doses lacking antibiotics, with no effect on reproductive output.ConclusionIn conclusion, artificial insemination (AI) with boar semen doses processed using Porcicoll, meeting modern requirements for alternatives to antibiotics, could be a promising state-of-the-art approach.

Semen preservation at 17 °C plays a pivotal role in the porcine breeding industry, though it presents challenges; not all ejaculates retain their quality during storage. Colloidal centrifugation has been developed to improve it since this method eliminates seminal plasma and microorganisms while simultaneously allowing the selection of spermatozoa based on gradient density centrifugation. This study aimed to evaluate whether two different colloid densities (1.026 g/ml - 20 %Porcicoll® and 1.104 g/ml - 80 % Porcicoll®) improve sperm bioenergetics, assessed using Agilent seahorse, along with key semen quality parameters such as viability, mitochondrial activity, oxidative stress and motility analyzed through flow cytometry and the CASA system over time. Fifteen ejaculates were divided into three groups: Control (CTR), 20 % Porcicoll® (P20), and 80 % Porcicoll® (P80). Sperm parameters were analyzed at days 0, 3, and 7 of storage. Results revealed a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis during storage, with a 15 % decrease in ATP production by day 3. P80-treated sperm showed higher ATP production on day 0 compared to P20. Sperm quality parameters such as viability and mitochondrial activity decreased after colloidal centrifugation, possibly because of early capacitation; we also hypothesize that SLC could increase sensitivity to the high glucose levels in the medium. Although Porcicoll® effectively selected metabolically active sperm, the lack of seminal plasma and limitations in media composition negatively affected long-term sperm quality. Further research is necessary to optimize media formulations and assess fertility outcomes of selected sperm for enhanced sperm preservation techniques.

Colloid centrifugation has several uses within spermatology. It has traditionally been used as a biomimetic method to select the most robust, fertile spermatozoa from a sperm sample. However, any sub-population can be selected, or the spermatozoa can be separated from seminal plasma, by changing the density of the colloid used. The colloids described here are silane-coated silica colloids in a buffered salt solution, optimized for different species. Although various other methods have been reported to select spermatozoa based on one or more physical properties, only colloid centrifugation is suitable for routine use on semen collection stations.Several variants of the method are available, depending on the volume of sample to be processed and the degree of separation required. All of them involve layering the sperm sample on top of the colloid in a suitably sized centrifuge tube, followed by gently centrifugation using a swingout rotor at 300 g for 20 min. After removal of the supernatant, the sperm pellet is resuspended in a suitable medium, depending on the intended use, e.g., semen extender for artificial insemination, cryomedium for sperm freezing, medium for in vitro fertilization. Thus, the method is practical for most semen collection facilities.

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation.