The objective was to compare equine semen parameters before and after filtration in a novel device based on microfluidics (VetCount™ Harvester). The hypothesis was that microfluidic-based filtration with the device improves semen parameters related to sperm quality.
Pilot prospective analyses including 55 Frozen-thawed stallion semen samples were performed. The semen samples evaluated originated from commercially frozen batches from Standardbred, Arabian and Warmblood stallions. The tested VetCount™ Harvester device contains a filter with 10μm micropores, and the filtration of the sperm cells across the filter is based on fluid dynamics and active movement. The device allows 1.0 mL of frozen thawed semen sample to be filtered into 0.8 mL of skim milk-based extender. The 55 samples were all filtered using the device. Concentration, percentage of Progressive Motile Sperm Cells (PMSCs), viability using propidium iodide, and percentage of morphological normal sperm cells was evaluated before and after filtration of the samples. Acrosome damage and DNA fragmentation analyses were performed on a smaller sample size (n=20). Sperm concentration and viability were evaluated using Nucleocounter® SP-100. Percentage of PMSCs and morphologically normal sperm cells, acrosome damage and DNA fragmentation were evaluated using CASA AndroVision®. Wilcoxon Signed-Rank tests were performed to assess statistical differences for sperm outcomes before and after filtration. Data is presented as median (25% and 75% percentiles). Significance was set at P<0.05. The median sperm concentration was 259 x 106/mL (176-368) before filtration and 18.4 x 106/mL (13-29.1) after filtration.
The following parameters improved significantly after filtration through the VetCount™ Harvester device: PMSCs (%) from 30% (12.9-36.6) to 76.0% (69.7-83.1) (p<0.0001), viability (%) from 38.1% (30.0-46.0) to 59.0 (47.0-71.0) (p<0.0001), morphologicallly normal sperm cells (%) from 52.0% (45.0-59.0) to 81% (76.0-86.0) (p<0.0001), acrosome damaged sperm cells (%) from 16.6% (11.3-20.2) to 2.5% (1.7-4.2) (p=0.0005), and sperm cells with DNA fragmentation from 6.38% (5.61-10.6) to 0.9% (0.6-1.42) (p<0.0001).
Recovered volume from the device was 0.7 mL. The extracted semen samples contained 14.6 % (8.7-22 %) of the Progressive Motile Sperm Cells in the native sperm samples. The total number of Progressive Motile Sperm Cells after filtration was 9.9 x 106 (5.8-15.9 x 106).
Percentage of PMSCs, sperm viability, sperm morphology, sperm with acrosome damage as well as sperm with DNA fragmentation can be significantly improved in a Frozen-thawed semen sample using the filtration technique in the microfluidic-based device. In practice, the device does not allow NC2022-002P filtration of dead and damaged sperm cells through the micropores in the filter. This leaves an improved semen sample to inseminate the mare without the negative impact of the dead and damaged sperm cells to the uterine environment.
The VetCount™ Harvester device is presented as a new technique that in the future could possibly improve the fertility of frozen equine semen samples used for artificial as well as it could improve the quality of a semen sample used for insemination or other assisted reproductive techniques such as Intracytoplasmic Sperm Injection (ICSI).
The aim of this study is to compare a novel sperm purification and capacitation technique based on microfluidics, called SwimCount™ Harvester, with density gradient centrifugation (DGC).
A pilot prospective study, that included sperm samples of 25 patients was conducted. Fresh ejaculate specimens and capacitated samples of each patient were analyzed according to WHO 2010 criteria with automatic image analysis. SwimCount™ Harvester and DGC were used to isolate sperm based on fluid dynamics and cell progressive motility. Fresh sperm samples and sperm resolved by both capacitation techniques were assessed for excessive histone retention indicating defective chromatin compaction and hence DNA maturation using aniline blue staining. Sperm chromatin fragmentation (SCF) of fresh and capacitated sperm samples was assessed by TUNEL on at least 20.000 sperm using flow cytometry. ANOVA test was performed in order to assess statistical differences between result variables.
Between March and April 2021, 25 men were recruited (mean age 34,7±8,7 years). Ejaculated sperm were assessed by automatic semen analysis, which yielded an average concentration of 60,1±31,8 x106/mL. After DGC and SwimCount™ Harvester, the sperm concentration was 13,2±9,0 and 13,3±10,0 x106/mL, respectively. The percentage progressive motile sperm of fresh sperm sample improved from 42,2±11,8% to 70,7±13,6% after DGC and 74,9±12,7% after Harvester (P<0,0001). Similar trends were obtained when morphology was analyzed. The percentage of normal sperm increased from 1,9±1,1%, for the raw samples, to 3,4±1,5 and 4,1±1,2% for the samples capacitated using DGC and microfluidics, respectively (P<0,0001). Another parameter, which benefited from the capacitation, was DNA maturation. The percentage of sperm DNA maturation increased from 65,3±7,4% to 72,5±7,3% when the samples were processed by DGC and 75,0±7,6% when the SwimCount™ Harvester was used (P<0,0001). In the same way, the percentage of live sperm increases from 74,0±7,9% in fresh sperm to 77,8±8,1% after DGC, and to 84,2±8,6% after using SwimCount™ Harvester (P<0,02). Although there are no significant differences, a decrease in SCF from 17,0±11,9% was observed in capacitated samples using DGC to 12,2±6,2% in raw samples (P>0,05). Nevertheless, after capacitation by SwimCount™ Harvester SCF fell to 8,2±7,3%, showing significant differences between both capacitation methods (P<0,006). The SwimCount™ Harvester showed statistically significant improved parameters concerning vitality and DNA fragmentation compared to density gradient centrifugation.
The SwimCount™ Harvester is presented as a new technique capable of automating the sperm capacitation process by microfluidics, minimizing variability between laboratories, human error and workload improving sperm parameters, especially concerning vitality and DNA fragmentation.
Fernando Meseguer, Rocío Rivera, Lorena Bori , Mª Ángeles Valera, Lucía Alegre, Marcos Meseguer.
1 IVI Foundation, Valencia (Spain), 2 IVIRMA Global, Valencia (Spain)