Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

The aim of this study was to develop and validate a novel, computer‐assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 28) with a haemocytometer using light microscopy, CASQ and computer‐assisted semen analysis (CASA; MMC sperm), following three independent dilutions. The limits of agreement between the haemocytometer and CASQ were ?13.1% to 13.8% and ?27.0% to 28.6% between the haemocytometer and CASA. The precision CVs (limits of agreement) were 5.7% (?7.8% to 8.9%) for the haemocytometer, 6.2% (?8.8% to 12.3%) for CASQ and 10.8% (?16.0% to 19.5%) for CASA. In Experiment B, spermatozoa were manually counted (n = 42) with the haemocytometer under fluorescent illumination using the CASQ sample. The limits of agreement between the CASQ and the haemocytometer were satisfactory (?4.6% to 4.6%) and the precision CVs (limits of agreement) were 6.2% (?9.0% to 11.4%) for the haemocytometer and 4.4% (?5.8% to 8.6%) for CASQ. The CASQ method was then clinically applied to compare the haemocytometer (light and fluorescent methods) with CASQ and CASA. Outlying data were removed. These studies demonstrated that CASQ was reliable and that the MMC sperm CASA was unreliable as methods for determining spermatozoal concentration in canine semen. Computer‐assisted spermatozoal quantification was also determined to be more precise than manual counting with the haemocytometer. Using the clinical protocol, the agreement between the haemocytometer and CASQ method was acceptable, but it was worse than in the experiments where duplicate samples and a larger volume of semen were analysed. The CASQ method may be a useful method to measure the membrane status of canine spermatozoa; however, further investigation is required. Counting spermatozoa using fluorescent microscopy and the haemocytometer may improve the efficiency of counting and the accuracy of the method.     

Effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars,analysed with a CASA‐Mot system

Motility is the most widely used indicator of sperm quality. Computer‐Assisted Semen Analysis (CASA) allows the objective evaluation of sperm motility parameters. CASA technology is a common tool to predict semen doses in farm animal reproduction. The kinds of video cameras used until now for image acquisition have presented limited frame rates (FR), which have a negative influence on the quality of the obtained data. The aim of the present work was to define the optimal frame rate for a correct evaluation of boar sperm motility and its subpopulation structure. Eighteen ejaculates from nine mature boars of the Pietrain breed were used. Using the ISAS^®v1 CASA‐Mot system, with a video camera working up to 200 Hz, six FRs (25, 50, 75, 100, 150 and 200 fps) were compared. ISAS^®D4C20 counting chambers, warmed to 37°C, were used. FR affected all the kinematic parameters, with curvilinear velocity (VCL) and BCF the most sensitive ones. All the parameters showed differences among animals. Non‐linear correlation showed the asymptotic level for VCL at 212 fps, being the highest FR for all the parameters. For future studies based just on progressive motility, almost 100 fps FR for 0.5 s must be used, while when kinematics must be considered, almost 212 fps for one‐second should be analysed. Three principal components were obtained (velocity, progressivity and oscillation), being similar at 50 and 200 fps. Cells were grouped in four subpopulations but with different kinematic and cellular distribution at both FRs.     

Method agreement between three different chambers for comparative boar semen computer‐assisted sperm analysis

The computer‐assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 10⁶ sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 μm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non‐progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

The comparison of two different extenders for the improvement of large‐scale sperm cryopreservation in common carp (Cyprinus carpio)

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large‐scale (elevated volume of sperm) freezing method in a controlled‐rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled‐rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.     

Economic evaluation of artificial insemination of sex‐sorted semen on a Brown Swiss dairy farm—A case study

Artificial insemination using sex‐sorted semen is employed to efficiently increase the number of female dairy calves born. Previous studies have determined that using sex‐sorted semen is beneficial to improve the management, but the mechanism by which it increases cattle numbers through objective indices of breeding remains unclear. This study focused on a Brown Swiss cattle herd in which frozen female sex‐sorted semen was systematically employed to increase the number of cattle. We analyzed the correlation between the increase in the number of cattle and the screening accuracy of sex‐sorted semen, measuring indices such as pregnancy rate and birth rate of female calves. Study revealed that: (1) production cost for female calves is influenced by the pregnancy rate, rate of female calves, and using sex‐sorted semen is less expensive than using nonsorted semen; (2) improvements in screening accuracy nearly doubled the number of cows and tripled the number of heifers in 5 years; and (3) use of sex‐sorted semen improved milk quality. The pregnancy rate was lower when sex‐sorted semen was used, but the birth rate of heifers was improved. Results suggest that artificial insemination using sex‐sorted semen is beneficial because it economically produces offspring to increase the herd.     

Effects of the supplementation with an high‐polyphenols extra‐virgin olive oil on kinetic sperm features and seminal plasma oxidative status in healthy dogs

The aim of this work was to evaluate the effects of the supplementation of two extra‐virgin olive oils (EVOO) having different polyphenols content, on canine spermatozoa kinetic parameters and seminal plasma oxidative status. The study was conducted on 12 clinically healthy dogs of different breeds (2–7 years, 5–48 kg of body weight) divided into two groups: an experimental group supplemented with EVOO (Coratina cultivar) high in polyphenols (H‐P) and a control group fed EVOO (Cima di Bitonto cultivar) low in polyphenols (L‐P). The oil was daily administered per os (1 ml/3 kg BW) before meal. Semen collection was made twice at 15 days distance (D0₁ and D0₂) and then at 30 (D30), 60 (D60) and 90 (D90) days. Semen concentration and kinetic parameters were measured using computer‐assisted sperm analysis (CASA) system to evaluate: sperm total count, sperm motile (MOT%), progressive motility (PROGR%) and its fractions, straight‐line velocity (VSL, μm/s), curvilinear velocity (VCL, μm/s), average path velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), straightness (STR%) and linearity (LIN%). On seminal plasma, reactive oxygen species (ROS) and biological antioxidant potential (BAP) were tested. From findings, no differences were found for sperm MOT, VSL, VCL, VAP, ALH, BCF, STR, LIN and BAP. A gradual enhancement of PROGR% was observed in H‐P group (p < .01). The ROS levels were higher in dogs H‐P compared to the other group (p < .05). In conclusion, our results highlight the positive effects of EVOO polyphenols on sperm PROGR% in healthy dogs.     

Red‐light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED‐based red light increases its reproductive performance following artificial insemination (AI). Red‐light stimulation with MaXipig^® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red‐light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red‐light stimulation. Our results indicate that a routine use of red‐light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurement of spermatozoal membrane status using computer‐assisted spermatozoal quantification (CASQ) and eosin‐nigrosin (EN) staining with manual counting after CFDA/PI staining. Analysis was performed on both fresh and thawed cryopreserved canine semen. Membrane‐disrupted spermatozoa (MDS) were counted using CASQ (n = 311) in an untreated sample and a completely membrane‐disrupted sample, and the percentage of membrane‐intact spermatozoa (MIS) calculated: (Total count ? Untreated sample count) ÷ Total count × 100. Spermatozoa were stained with a one‐step EN stain (n = 501), and then, at least 100 spermatozoa were manually examined under ×1,000 magnification and classified as MDS (stained with eosin) or MIS (non‐stained). Spermatozoa from the same samples were also stained with CFDA/PI, and then, at least 200 spermatozoa were manually examined under ×1,000 magnification and classified as MIS (completely stained by CFDA) or MDS. The percentage of progressively motile spermatozoa (PMS) was determined by both computer‐assisted semen analysis (CASA) and subjective methodologies, and the data were subsequently analysed to measure the agreement between the CASQ and EN methods with the CFDA/PI technique using Bland–Altman methodology. Pearson's correlation was measured between the MIS and PMS percentage samples and correlation coefficients compared. The mean MIS percentage was lower for CASQ and higher for EN than in CFDA/PI for all comparisons. The agreement of MIS percentage between CASQ and CFDA/PI was ?20.2% to 32.0%, and between EN and CFDA/PI was ?32.9% to 14.9%. In all methods, the MIS and PMS percentages were correlated (p < .001). Measurement of CFDA/PI appeared to be the most reliable and accurate method of determining MIS percentage in dogs. Further investigation is required to determine whether the CASQ technique can be improved. Eosin‐nigrosin staining also appeared to be unreliable at MIS <80% and overestimated the MIS percentage.