Colloid Centrifugation Research Articles

In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism

BackgroundConservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24–48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. Material and methodsProcessed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca2+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. ResultsAfter 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca2+ increased in all treatment groups compared to time 0h. Discussion and conclusionViable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.

Strategies to Reduce the Use of Antibiotics in Fresh and Chilled Equine Semen.

The study assessed the impact of four equine semen processing techniques on sperm quality and microbial load immediately post-processing and after 48 h of refrigeration. The aim was to explore the potential reduction of prophylactic antibiotic usage in semen extenders. Semen from ten adult stallions was collected and processed under a strict hygiene protocol and divided into four aliquots: Simple Centrifugation with antibiotics (SC+), Simple Centrifugation (SC-), Single-Layer Colloidal Centrifugation (CC-), and Filtration (with SpermFilter®) (F-), all in extenders without antibiotics. Sperm motility, viability, and microbial load on three culture media were assessed. No significant differences were observed in the main in the sperm quality parameters among the four protocols post-processing and at 48 h (p < 0.05 or p < 0.1). Microbial loads in Columbia 5% Sheep Blood Agar and Schaedler vitamin K1 5% Sheep Blood Agar mediums were significantly higher (p < 0.10) for raw semen than for CS+, CC-, and F- post-processing. For Sabouraud Dextrose Agar medium, the microbial load was significantly higher (p < 0.10) in raw semen compared to CS+ and F-. No significant differences (p < 0.10) were found in 48 h chilled samples. Regardless of antibiotic presence, the evaluated processing methods, when combined with rigorous hygiene measures, maintained semen quality and reduced microbial load to the same extent as a traditional protocol using antibiotics.

The efficacy of various protocols of colloidal centrifugation of bovine sperm for IVF

The article presents data on the effectiveness of using various sperm preparation protocols by colloidal centrifugation (density gradient centrifugation method) before procedures of assisted reproductive technologies, including in vitro artificial insemination. The use of the method can affect the quality of sexual gametes and the viability of embryos, increase the percentage of successful embryotransplantation and contribute to the appearance of highly productive offspring.As a result of the research, the most optimal protocol of centrifugation by the method of gradient layering with indicators of the first and second centrifugation 700g – 20 min and 100g – 10 min, respectively, was determined by morphofunctional characteristics. The average rate of motile spermatozoa was 84.1±5.1%, progressive spermatozoa – 68.7±5.1%, the rate of rectilinear translational motion was 49.69 microns/sec. Morphology and viability meet quality standards.

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Selection of frozen-thawed semen for standard in vitro fertilization

Since the report of a repeatable method for standard in vitro fertilization in the horse (IVF) using prolonged sperm pre-incubation (Felix et al., Biol. Reprod. 107:1551-1564, 2022), efforts have been directed toward applying the IVF procedure clinically. A major barrier to clinical use is the need for fresh semen. In this study, we compared three techniques – swim-up (SU), colloid centrifugation (CG), and microfluidics (MFL) – to select frozen-thawed sperm for standard IVF. Media were modified TALP (TALP-R and FT-PHE, Felix et al., 2022). Straws of frozen semen from one fertile stallion were used. For SU, 200 µL frozen-thawed semen was layered under 1 mL FT-PHE in each of two 5-mL tubes. The tubes were incubated for 30 min in 5% CO2 in air, then the uppermost 750 µL of medium was collected, combined, centrifuged and the pellet resuspended in TALP-R to ∼10 million/mL. The suspension was added to 45-µL droplets of FERT-TALP to a concentration of 1 million sperm/mL (in 50 µL) and the droplets were incubated in 5% CO2 in air. For CG, frozen-thawed semen (400µL) was layered on 3 mL of EquipureTM colloid and centrifuged for 20 min at 200g. The sperm pellet was washed once in FT-PHE, then resuspended in TALP-R and sperm added to droplets as above. For MFL, 400µL frozen-thawed semen was combined with 500µL of FT-PHE andthis suspension was placed into the loading chamber of a commercial microfluidic device. FT-PHE (700µL) was placed into the collection chamber. The device was incubated for 30 min in 5% CO2 in air, during which time sperm migrated through a porous membrane into the collection chamber. After incubation, 500µL fluid from the collection chamber was combined with 500µL FT-PHE then centrifuged for 5 minutes at 300g. The pellet was resuspended with TALP-R and sperm added to droplets as above. After droplets were incubated for 10h, in vitro-matured equine cumulus-oocyte complexes (COCs) were added. After 3h co-incubation, the COCs were transferred to embryo culture medium for 32h, then denuded and stained with DAPI. Cleaved embryos with two or more normal nuclei were considered to represent fertilization. Sperm selection by MFL resulted in a higher cleavage rate per co-cultured oocyte (23/32, 71.9%) than did the other two selection methods (8/35, 22.9% and 5/31, 16.1% for SU and CG, respectively). We conclude that standard equine IVF can be achieved with frozen-thawed semen and that sperm selection using a microfluidic device is associated with a higher cleavage rate than is selection using swim-up or colloid centrifugation.

Epididymal stallion sperm quality improves with single layer centrifugation prior to cryopreservation or post-thawing

Cryopreservation of epididymal spermatozoa is the last method for preserving gametes of valuable stallions in case of sudden death, severe injuries, or orchiectomy. Due to harvesting methods, sperm is unavoidably contaminated with epithelial and red blood cells, leukocytes, debris, and bacteria. These contaminants and dead spermatozoa can cause detrimental effects on sperm quality. Colloid centrifugation can reduce the load of these contaminants and select a subpopulation of motile spermatozoa. The objective of this experiment was to evaluate whether single layer centrifugation (SLC) prior to cryopreservation or post-thawing improves the quality of stallion epididymal sperm. Ten stallions were submitted to bilateral orchiectomy and harvesting of epididymal cauda spermatozoa (n=20) was performed by retrograde flushing with an extender. Epididymal sperm samples were subjected to conventional centrifugation (no colloid - 600xg for 20 minutes), and SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). SLC was performed at 300 x g for 10 minutes (Androcoll-Equine, Minitube, Tiefenbach, Germany). All samples were cryopreserved, thawed, and evaluated. The post-thaw group (SLC+) was thawed, centrifuged by SLC, and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility and progressive motility were evaluated with computer-assisted semen analyses (CASA). Sperm morphology was evaluated by examining a wetmount preparation of unstained samples fixed in formol saline. Mitochondrial functionality, membrane integrity, and DNA integrity were evaluated in a fluorescence microscope with specific fluorescent dyes. All variables were normally distributed according to the Shapiro–Wilk test. Data were evaluated by descriptive statistics, one-way analysis of variance, and Tukey's test. Significance was set at p <0.05. SLC-PC (24.6 ± 3.6%) and SLC+F (26.2 ± 4%) treatments yielded significantly higher total sperm motility than SLC+C (16.7 ± 1.7%) and conventional (16 ± 3.5%) treatment. SLC+F (17.5 ± 3.8%) yielded significantly higher progressive motility than the conventional (8.8 ± 2%), SLC+C (9.8 ± 1.3%), and SLC-PC (12.9 ± 4.1%) treatments. The mitochondrial functionality of all SLC groups was significantly higher (SLC-PC 72 ± 2%; SLC+C 74.6 ± 1.8%; SLC+F 82.5 ± 1.9%), than that of the conventional group (25.2 ± 2.1%). Plasma membrane integrity was higher in SLC-PC (48.6 ± 4%) than in SLC+F (30 ± 6.5%), SLC+C (36 ± 4.8%), and conventional (22.8 ± 4.5%) samples. DNA integrity and percentage of morphologically normal samples were unaffected by treatment. SLC is a simple procedure that can be performed prior to cryopreservation or post-thawing and that can improve stallion epididymal sperm quality. Epididymal spermatozoa submitted to SLC only after thawing is best extended using freezing extender than cooling extender.

Colloid centrifugation as an alternative to antibiotics in stallion semen preparation

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

The effect of sex-sorting on stallion semen quality

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.

Evaluation of simple strategies to reduce the use of antibiotics in equine fresh sperm

According to the World Health Organization in 2021, the development of antimicrobial resistances is considered one of the top ten global public health threats. Microbial load negatively affects sperm quality and preservation (Ortega-Ferrusola and Peña, Reproduction in Domestic Animals. 2009; 44(3):518-522), which implies the use of antibiotics in semen extenders. The objective of this study was to study the effect of three semen processing techniques on quality and microbial load of fresh equine sperm to evaluate the possibility of reducing or eliminating the prophylactic use of antibiotics in the future. Ten adult stallions were included (one ejaculate per male). To avoid environmental contamination during sperm collection and processing, a protocol that maximizes hygiene measures was implemented. Aliquots of each ejaculate were processed following 4 protocols: Simple Centrifugation (450g x 7 min) in extender with antibiotics (SC+, as control group), Simple Centrifugation in extender without antibiotics (SC-), Single-Layer Colloidal Centrifugation (300g x 20 min) in extender without antibiotic (CC-) and Filtration (SpermFilter®) in extender without antibiotics (F-). Sperm motility (total and progressive motility, TM and PM, respectively, %), viability (Alives, %), and microbial load (on three different culture media (Columbia 5% Sheep Blood Agar, COS, general purpose medium for non-fastidious and fastidiousmicroorganisms, Sabouraud Dextrose Agar, SDA, for yeasts and molds, and Schaedler vitamin K1 5% Sheep Blood Agar, SCH, for fastidious anaerobic bacteria) were evaluated after processing. Statistical analysis was performed by Kruskal-Wallis, ANOVA and post hoc tests (p<0.05). Results are expressed as mean values ± SD. No significant differences were found between the control (SC+) and other groups (SC-, CC- and F-), neither in sperm characteristics (61,32a ± 20,84, 59,13a ± 21,84, 63,19a ± 18,87 and 52,54a ± 21,08 for PM and 84,60a ± 9,21, 85,05a ± 9,14, 84,90a ± 13,5 and 77,35a ± 13,27 for ALIVES respectively) nor in microbial load (216,50a ± 504,41, 348,30a ± 792,99, 731,30a ± 2180,11 and 51,70a ± 64,64 for COS; 1,00a ± 3,16, 11,00a ± 31,43, 4,00a ± 6,99 and 1,00a ± 3,16 for SDA and 25,00a ± 57,59, 41,00a ± 60,45, 10,00a ± 18,26 and 26,00a ± 42,48 for SCH respectively). The present processing methods, in combination with management measures that prevent environmental contamination, maintained sperm quality and microbial load of equine fresh semen to the same extent as traditional sperm processing protocols with antibiotics. Acknowledgements: Authors thank P. Cuesta for statistical assistance. This research was funded by Madrid Institute for Rural, Agricultural and Food Research and Development (IMIDRA).

Storing stallion sperm in SpermSafe™ at 17°C may improve fertility by reducing mPTP formation

Stallions with sperm that do not tolerate cooling have limited commercial potential. Cooling increases sperm intracellular calcium (Ca2+i; White, Reproduction, Fertility and Development. 1993;5:639-58), which in human spermatozoa triggers formation of the mitochondrial permeability transition pore (mPTP), leading to apoptosis (Treulen et al., Human Reproduction. 2015;30:767-76). However, the mechanism of sperm mPTP formation is yet to be described in the horse. As such, this study investigated whether mPTP formation occurs in stallion spermatozoa following Ca2+i increase and whether storing spermatozoa at higher temperatures can minimize Ca2+i increase and mPTP formation, thereby improving fertility. This would be investigated by alternatively storing sperm at 17°C in SpermSafe™ (BREED Diagnostics), a medium designed for liquid-storage at higher temperatures. Experiment 1 utilised three ejaculates from each of three pony stallions (n=9) to investigate the relationship between Ca2+i and mPTP formation. Semen was collected using an artificial vagina (AV) and extended 2:1 (extender:semen) using EquiPlus (Minitube). Spermatozoa were isolated from seminal plasma and extender using single-layer colloidal centrifugation before being resuspended to 20 × 106/ml in Biggers, Whitten, and Whittingham medium. Spermatozoa were pre-loaded with calcein-AM (fluorescent mPTP probe) and exposed to various doses of ionomycin to induce the Ca2+i increase. Data were checked for normaldistribution (Shapiro-Wilk test), with oneway ANOVA (treatment effect) and Dunnets test (comparing treatments and the control) used to identify significant differences, and ‘stallion’ used as a blocking term. The mPTP formed (loss of calcein fluorescence) following ionomycin-induced Ca2+i increase at 5 nM (100±16.4 AFU vs. 44.3±8.3 AFU for control and 5 nM ionomycin, respectively; P≤0.001), indicating that stallion spermatozoa do undergo mPTP formation in response to elevated Ca2+i. Experiment 2 utilised a fertile (83% per-cycle conception rate [PCCR] with fresh semen; n=6), 5-year-old Warmblood stallion with a history of poor fertility using cooled semen. Semen was collected and extended as described above and was washed via cushioned centrifugation. Sperm pellets were resuspended to 50 × 106/ml in either EquiPlus (cooled in a standard commercial shipper) or in SpermSafe (stored at 17°C in an EquOcyte shipper) for 24 h. Due to the logistical constraints of commercial practice, split ejaculates could not be utilized, so each sample was a separate ejaculate. Fixed-time AI was performed 24 h following semen collection and ovulation induction. Pregnancy rates of 0% (0/6) and 67% (8/12) were achieved following AI with cooled vs. SpermSafe-stored spermatozoa, respectively (Chi Square; P≤0.01). In conclusion, these data suggest that storing stallion sperm at 17°C in SpermSafe before AI may improve fertility in stallions with poor cooling tolerance, potentially due to reduced mPTP formation.

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

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

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection.

Optimization of the Equine-Sperm Freeze Test in Purebred Spanish Horses by Incorporating Colloidal Centrifugation.

The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations for equine-sperm cryopreservation. This means that artificial insemination with frozen semen from these stallions is less widespread than in other breeds. In this study, we investigated if the incorporation of single-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing, observing the influence of centrifugation and freezing extender protocol on post-thawed sperm motility. Using colloidal centrifugation, the percentage of ejaculates available to be frozen was increased from 35% (6/17) to 71% (12/17), doubling the number of samples that could have been subjected to cryopreservation. We only found significant differences in linearity (LIN) and lateral head displacement (ALH) after 5 min of incubation at 37 °C between colloidal and simple centrifugation processing techniques. No significant differences were found between the two different colloidal protocols in any of the variables considered. Colloidal centrifugation allowed us to obtain, from worse fresh-quality ejaculates, thawed sperm doses with similar quality to that of good-quality ejaculates. BotuCrio® produced, in general, higher motility parameters and its characteristics than the other extenders analyzed, with significant differences found in comparison to Inra-Freeze® and Lac-Edta in both total (MOT) and progressive motility (PMOT) when using colloidal centrifugation and only in PMOT when applying simple centrifugation. Colloidal centrifugation optimized the efficiency of cryopreservation, as it allowed us to increase the number of ejaculates of Purebred Spanish Horses suitable to be frozen. Including these semen processing techniques in the freeze test could help to optimize equine-sperm cryopreservation protocols, especially when dealing with individuals or breeds for which initially low sperm quality prevents or limits their inclusion in sperm cryopreservation programs.

Sperm Quality in Young Bull Semen Can Be Improved by Single Layer Centrifugation.

Simple SummaryGenomic selection enables bulls with desirable genes to be identified early in life. Livestock producers need to use the semen from young bulls as early as possible for efficient milk and meat production with fewer greenhouse gas emissions. However, semen from young bulls is often of lower quality than needed for freezing for commercial artificial insemination. Colloid centrifugation selects spermatozoa with the desirable characteristics needed for fertilization from the rest of the ejaculate. In this study, split ejaculates from young bulls were prepared with or without colloid centrifugation. Using this technique, sperm doses of acceptable quality for artificial insemination could be produced from ejaculates that would otherwise be discarded. Thus, the semen from young bulls would be usable for artificial insemination sooner than is currently the case.Interest in using semen from young bulls is increasing due to identifying promising animals by genomic selection. However, sperm quality in these ejaculates may not reach currently accepted standards for the cattle breeding industry. The purpose of this study was to determine if centrifugation of semen from young bulls through the Bovicoll colloid could improve sperm quality sufficiently for the frozen semen to be acceptable for artificial insemination. Ejaculates from 19 young bulls were split and either processed by Single-Layer Centrifugation (SLC) or not (CON) before freezing. After thawing, sperm quality was evaluated by determination of membrane integrity, mitochondrial membrane potential, DNA integrity, production of reactive oxygen species, sperm morphology and motility. Approximately half of the CON samples reached acceptable post-thaw quality (membrane integrity ≥ 40%) despite being below the breeding company´s desired sperm concentration threshold pre-freezing. In the remaining samples, sperm quality was improved by SLC such that 45% of them reached acceptable quality post-thaw. Almost 75% of the young bull sperm samples could have produced usable frozen semen doses by adjusting the breeding company´s current processing protocols. Since lowering the generation interval has a direct effect on the genetic gain per year, SLC could aid genetic progress in cattle breeding.

Filtration techniques are advantageous over colloidal centrifugation in improving freezability of low-quality buffalo bull (Bubalus bubalis) ejaculates

The study compared efficacy of three sperm selection techniques in improving freezability of low-quality Murrah buffalo bull ejaculates. Sephadex (SEP), Sephadex ion-exchange filtration (SIE), and 40/80% BoviPure ™ (BP) gradient centrifugation protocols were standardized (ejaculates, n = 24). In Experiment-I, Sephadex G-75, G-100, and combined Sephadex G (75–100) column filtrates were compared. In Experiment-II, BP protocols: 200 g-10 min, 250 g-5, and 10 min, 300 g-10, and 15 min were compared. In fresh semen, Sephadex G (75–100) filtration and 250 g-5 min BP protocol improved sperm functions and were used in Experiment-III, where SEP G (75–100), SIE G (75–100), and 250 g-5 min BP processed ejaculates (n = 48) were cryopreserved and compared at post-thaw stage. The mean recovery rate differed in order: SEP > SIE > BP. SIE filtration significantly improved progressive motility, livability, membrane integrity, bovine cervical mucus penetration and live non-apoptotic sperm. Compared with control, all three techniques equally reduced post-dilution and post-thaw lipid peroxidation (LPO) rate. SEP post-thaw filtrates observed lower cryocapacitation-like changes, LPO (C11‐BODIPY581/591), and higher active mitochondria than other treatments. SIE and SEP equally improved post-thaw acrosome-intact sperm over BP. Filtration techniques, preferably, Sephadex ion-exchange filtration can most efficiently process low-quality buffalo bull ejaculates for cryopreservation and improve freezability.

Modern technologies for storing semen of domestic animals without the addi-tion of antibiotics

Currently, there is a trend of breeding domestic animals through artificial insemination. As a result, very large amounts of sperm diluents containing antibiotics are used in animal husbandry. Antimicrobials are added to the semen diluent to control the growth of bacteria that contaminate the semen during selection. The proportion of antibiotic-resistant bacteria is steadily rising, threatening the entire health care system. That is why all fields of antibiotics application face the task of finding alternatives to this approach. The purpose of our study was to systematize modern technologies and methods of storing domestic animals’ semen which could reduce or eliminate the use of antibiotics, and would be an important step in the fight against multidrug-resistant bacteria. Due to the negative impact of antibiotics on sperm quality and their fertilizing ability, new alternative methods for sperm storage are constantly being improved and developed. The most common are low-temperature storage, physical methods to reduce bacterial stress, the use of antimicrobial peptides, nanoparticles and the use of various substances of animal, plant or other origin. The possibility of boar sperm low-temperature storage may open up completely new approaches in the future by optimizing the cooling rate. Colloidal centrifugation as one of the physical methods is a practical means of reducing the bacterial load in sperm samples and it can be effectively used applying equipment that is available at many breeding plants. Antimicrobial peptides or nanoparticles of iron oxide may be a useful alternative to the addition of antibiotics during sperm storage. Antimicrobial peptides have been shown to control the growth of aerobic and anaerobic bacteria in relatively low concentrations without adversely affecting sperm quality and fertility. However, it is substantiated that nanoparticles with the size of 40 – 60 nm have significant antimicrobial ability against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. At the same time, further studies are needed on the use of various substances of animal or plant origin (royal jelly, aloe vera, algae extracts), as well as determining adequate concentrations of these new compounds that should be effective in fighting bacteria and not affect quality characteristics of sperm.

Comparison of single layer centrifugation and magnetic activated cell sorting for selecting viable boar spermatozoa after thawing

Sperm selection techniques, such as magnetic activated cell sorting (MACS) and colloid centrifugation, are reported to select good quality spermatozoa from semen samples of various species. Although the sperm quality of fresh boar semen is usually good, cryopreservation has a negative effect on parameters such as plasma membrane integrity and mitochondrial activity. Therefore, the objective of the present study was to determine whether MACS or centrifugation through a single layer of colloid (Single Layer Centrifugation, SLC) would be beneficial in enriching thawed boar sperm samples for viable spermatozoa with active mitochondria and good chromatin integrity. Frozen samples from three boars, three ejaculates per boar, were thawed and split. One part was selected by MACS, one was prepared by SLC, and the remainder served as the control. Controls and the selected sperm samples were evaluated for sperm quality (plasma membrane integrity, chromatin integrity, mitochondrial membrane potential and production of reactive oxygen species). Although several aspects of sperm quality were improved in the SLC-selected sperm samples compared to control, the flow-through MACS samples were only improved in having a lower proportion of spermatozoa with immature chromatin (Hi green fluorescence) compared to the labeled control. Sperm quality in the SLC samples was better than in the flow-through samples from MACS. Therefore, despite promising reports of the use of MACS for selecting good quality spermatozoa from semen in other species, the method was not useful for improving sperm quality in the thawed boar sperm samples in this experiment.

Antimicrobial Resistance in Equine Reproduction.

Simple SummaryBacteria can develop resistance to antibiotics, resulting in the appearance of infections that are difficult or impossible to treat. This ability enables bacteria to survive in hostile environments and can result from exposure to even small amounts of antibiotic substances. Bacteria are present in the reproductive tract of the horse; they can develop resistance to antibiotics, because the animal has been treated for an infection, or due to insemination with a semen dose that contains antibiotics. Bacteria colonize the membrane lining the male reproductive tract and are transferred to the semen during collection. They can cause sperm quality to deteriorate during storage or may cause an infection in the mare. Therefore, antibiotics are added to the semen dose, according to legislation. However, these antibiotics may contribute to the development of resistance. Current recommendations are that antibiotics should only be used to treat bacterial infections and where the sensitivity of the bacterium to the antibiotic has first been established. Therefore, adding antibiotics to semen extenders does not fit these recommendations. In this review, we examine the effects of bacteria in semen and in the inseminated mare, and possible alternatives to their use.Bacteria develop resistance to antibiotics following low-level “background” exposure to antimicrobial agents as well as from exposure at therapeutic levels during treatment for bacterial infections. In this review, we look specifically at antimicrobial resistance (AMR) in the equine reproductive tract and its possible origin, focusing particularly on antibiotics in semen extenders used in preparing semen doses for artificial insemination. Our review of the literature indicated that AMR in the equine uterus and vagina were reported worldwide in the last 20 years, in locations as diverse as Europe, India, and the United States. Bacteria colonizing the mucosa of the reproductive tract are transferred to semen during collection; further contamination of the semen may occur during processing, despite strict attention to hygiene at critical control points. These bacteria compete with spermatozoa for nutrients in the semen extender, producing metabolic byproducts and toxins that have a detrimental effect on sperm quality. Potential pathogens such as Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa may occasionally cause fertility issues in inseminated mares. Antibiotics are added during semen processing, according to legislation, to impede the growth of these microorganisms but may have a detrimental effect on sperm quality, depending on the antimicrobial agent and concentration used. However, this addition of antibiotics is counter to current recommendations on the prudent use of antibiotics, which recommend that antibiotics should be used only for therapeutic purposes and after establishing bacterial sensitivity. There is some evidence of resistance among bacteria found in semen samples. Potential alternatives to the addition of antibiotics are considered, especially physical removal separation of spermatozoa from bacteria. Suggestions for further research with colloid centrifugation are provided.

Non-enzymatic extraction of spermatozoa from alpaca ejaculates by pipetting followed by colloid centrifugation

Viscous camelid ejaculates present problems for sperm handling and sperm preservation. In the present study, a technique that had been used for dromedary camel semen was tested with alpaca semen. Ejaculates (n=9) were collected by artificial vagina at San Marcos University, Lima, and were liquefied by gentle pipetting in tris-citrate-fructose. Half of the sample was prepared by Single Layer Centrifugation (SLC) through a colloid; the other half was centrifuged without colloid as a control. Each control and SLC sample was then split into two parts; one part was stored cooled for 24 h at 5 °C and the other part was frozen, resulting in 4 treatments for each ejaculate. All samples were evaluated for sperm motility, hypoosmotic swelling test (HOST), plasma membrane integrity, and morphology, immediately after centrifugation and again after storage Total motility and plasma membrane integrity were greater in samples prepared by SLC than controls (motility 72±13% vs. 57±7%; plasma membrane integrity 63±13% vs. 54±8%, for SLC and controls respectively). Normal morphology and HOST were not different between treatments (65±13 vs. 61±13% and 42±6 vs. 39±10%, for SLC and controls respectively). After 24 h cooled storage, motility and plasma membrane integrity were greater for SLC samples (motility: 51±16 vs. 34±15%; p<0.001; membrane integrity: 51±15 vs. 40±18%; P < 0.05 for SLC and controls, respectively); HOST (40±14 vs. 34±11%) and normal morphology (67±13 vs. 63±14%) were not different between treatments. Sperm quality decreased considerably after cryopreservation (P<0.001 for all parameters); however, motility (P<0.01), plasma membrane integrity (P<0.05) and morphology (P<0.05) were higher for SLC than for controls. These results indicate that alpaca spermatozoa can be extracted from semen using a combination of pipetting and SLC, potentially with a beneficial effect on sperm quality. Samples could be stored cooled for 24 h, retaining better motility than controls; motility and plasma membrane integrity were greater in SLC samples than controls after freezing and thawing but the freezing protocol requires improvement.

Отработка и исследование процесса получения фотонно-кристаллических пленок методом центрифугирования

The article describes one of the promising methods of depositing highly ordered arrays of polystyrene microspheres from colloidal suspension through centrifugation. The simplicity and availability of the method determine the sensibility of its application in the deposition of high-quality ordered photonic-crystal planar structures for devices in nanophotonics, nanoelectronics, sensorics and security systems. A theoretical description of the process of colloidal microspheres self-organization into an ordered structure, the so-called opal matrix, occurring during centrifugation of a suspension is given. The technological equipment and accessories required for the implementation of the method in laboratory conditions are shown. The technological factors affecting the reflection of the formed structures in the region of the photonic band gap have been studied; a mathematical model of the colloidal suspension centrifugation process was developed and analyzed. The main modes of the centrifugation process ensuring an acceptable quality of the samples have been selected.