Effect Of Single Layer Centrifugation Research Articles

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.

Colloid centrifugation reduces bacterial load in chilled dog semen

Conventional semen extenders contain antibiotics to prevent bacterial growth. Finding alternatives would be beneficial to minimize the development of bacterial resistance mechanisms. The aim of this study was to determine the effect of Single Layer Centrifugation (SLC) with Canicoll of dog semen on microbial load and sperm quality during cooled storage. Twenty-four ejaculates were obtained from healthy dogs by digital manipulation. Samples were diluted in Tris-citrate-fructose extender without antibiotics and divided into two treatment groups: SLC-selected samples and unselected samples. Sperm motility (CASA), viability and acrosome integrity (PI/FITC-PNA) as well as bacterial load of each microorganism species (colony-forming units/mL) were assessed at 0 and 48 h of storage at 4 °C. Results indicate SLC-selected dog spermatozoa have greater percentages of motility, viability and acrosome integrity (P < 0.05). Bacterial growth in SLC sperm samples was less (P < 0.05) than unselected samples. Removal of individual bacterial species varied from 91 % to 98 % for Escherichia coli (91.62 %), Streptococcus spp. (98.18 %), Staphylococcus spp.(95.33 %) and Pseudomonas spp. (92.50 %). In conclusion, the use of SLC with Canicoll has the potential to decrease bacterial load in chilled dog semen.

8 Effect of Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen on Plasma Membrane Stability and Lipid Peroxidation After Cryopreservation

The discontinuous density gradient with Percoll® is routinely used for sperm selection before IVF in bovine in vitro embryo production. However, few studies have been addressed to investigate the use of this colloid in single layer centrifugation (SLC) before sperm freezing. Therefore, this study aimed to verify the effect of SLC with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing on plasma membrane stability (PMS) and lipid peroxidation (LP) of frozen–thawed bull semen. Three Nellore bulls housed at the Artificial Insemination Station were used. The ejaculates, 6 of each bull, were collected by artificial vagina. On the day of each collection, semen was first assessed for sperm motility, concentration, and morphology. Then, the ejaculates were pooled and ~1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (without glycerol) or undiluted (UN) was placed on top of a 9-mL column of PP at concentrations of 70% or 90%, resulting in the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control group, the supernatant was removed and the pellet of spermatozoa was resuspended in freezing extender (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Then, 0.5-mL straws were filled, cooled, and frozen. Sperm samples of each group and different days of collection were thawed in a water bath at 37°C for 30 s and diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (Sigma Co., St. Louis, MO, USA). Then, samples were stained for PMS with the association of MitoSoxRed (Molecular Probes Inc., Eugene, OR, USA) and YO-PRO-1 (Thermo Fisher Scientific, Hampton, NH, USA) and with C11-BODYPY (Thermo Fisher Scientific) for LP. The sperm samples were analysed by flow cytometer (BD LSR, Fortessa, Becton Dickinson, Mountain View, CA, USA), with the data analysed through use of BD FACSDIVATM software (version 6.1). Analysis of variance and Tukey’s test were used for statistical analysis with P &lt; 0.05 taken as significant. A lower percentage of cells displaying no destabilised membrane was found in 70D (27.6 ± 5.6) compared with the control group (43.1 ± 3.0), but no difference was observed among control group, 70UD (37.2 ± 5.1), 90UD (42.2 ± 7.5), and 90D (32.3 ± 6.5). There was a tendency (P = 0.08) for a higher proportion (P &lt; 0.05) of cells showing LP in control group (48.2 ± 5.7), 70D (48.3 ± 8.5), and 90D (44.43 ± 7.3) compared with the undiluted groups. Thus, spermatozoa selection using SLC with Percoll Plus® did not enhance membrane stability, but sperm LP might be diminished by using SLC with lesser dilution or concentration. It is suggested that lower generation of reactive oxygen species occurred when SLC with PP was used, indicating a possible protective effect on membrane phospholipids. Further studies, including anion superoxide, hydrogen peroxide, and mitochondrial membrane potential could elucidate these findings. This research was supported by FAPESP (grant #2015/20986-3), Tairana AI Station and Master Fertility, Brazil.

10 Evaluation of Frozen Sperm Quality After Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen

The single layer centrifugation (SLC) with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing is not a common technique used for selection of spermatozoa in bovine. Thus, this study aimed to verify the effect of SLC with PP before freezing on integrity of plasma and acrosome membranes (IPAM), phospholipid translocation (PT), and mitochondrial membrane potential (MMP) of frozen–thawed bull sperm. Three Nellore bulls housed at the Tairana Artificial Insemination Station were used. The ejaculates (6/bull) were collected by artificial vagina and assessed for sperm motility, concentration, and morphology. Then, the sperm were pooled and ~1 billion spermatozoa, either diluted [D; 1:2 (v/v)] in freezing extender (FE; tris, fructose, citric acid, egg yolk and antibiotics, without glycerol) or undiluted (UN), were placed on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90%, to form the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control (C), the supernatant was discarded and the pellet diluted in FE (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Afterward, 0.5-mL straws were filled, cooled for 5 h at 4°C, and frozen in a programmable freezer (Digitcool, IMV, L’Aigle, France) following the temperature/time curve: from 4°C to –10°C (5°C min−1), –10°C to –100°C (40°C min−1) and from –100°C to –140°C (20°C min−1), in a total of 8 min, when the straws were plunged into and stored in liquid nitrogen until evaluation. Thawed sperm (at 37°C/30 s) was diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (100 μg mL−1; Sigma Co., St. Louis, MO, USA). After that, samples were stained for membrane integrity with the association of fluorescent probes propidium iodide (PI, 50 μg mL−1; Sigma Co.), fluorescein thiocyanate (FITC)-Pisum sativum agglutinin (PSA, 1 mg mL−1; Sigma Co.) and Annexin V-APC (BD Pharmingen, Franklin Lakes, NJ, USA), and with MitoStatus Red (20 nM; BD Pharmingen) and YO-PRO-1 (7.5 μM; Molecular Probes Inc., Eugene, OR, USA) for MMP. Sperm samples were analysed by flow cytometer (BD LSR; Fortessa, Becton Dickinson, Mountain View, CA, USA) and the results expressed as percentage of intact cells or qualitative fluorescence expressed in arbitrary units (AU). Analysis of variance and Tukey’s test were used for statistical analysis with P &lt; 0.05 taken as significant. There were no differences between groups for IPAM (values ranging from 45.9 ± 7.0% to 55.6 ± 8.5%). Similarly, results of PT translocation did not differ among the groups (range from 34.7 ± 7.0% to 47.6 ± 7.0%). However, there was a tendency of increasing MMP (P = 0.10) in 70UD (1789 ± 258 UA), 70D (1776 ± 162.1 UA), and 90UD (1757 ± 133.8 UA) compared with C (1368 ± 267.4 UA) and 90D (1356 ± 145 UA). In conclusion, SLC did not compromise sperm membrane functionality and it seemed to select spermatozoa with higher mitochondrial functional activity when centrifuged at the concentration of 70% and 90D. This research was funded by FAPESP # 2015/20986-3, Tairana Artificial Insemination Station, MasterFertility Ltda, Brazil.

54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P &lt; 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P &lt; 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P &lt; 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

Effect of single layer centrifugation on motility of epididymal stallion sperm – preliminary results

Effect of single layer centrifugation on motility of epididymal stallion sperm – preliminary results

Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen.

Additional means are needed for evaluating the quality of stallion spermatozoa in semen doses for AI. Mitochondrial membrane potential (ΔΨm) has been linked to fertility in some species, but is rarely used in the evaluation of cooled stallion semen; metabolic activity may be associated with reactive oxygen species production (ROS). In the present study, ΔΨm and ROS production were measured in doses of cooled stallion semen. The effect of colloid centrifugation on these parameters was also investigated. In this case, colloid centrifugation involves centrifuging a sperm sample through a silane-coated silica colloid formulation to retrieve the most robust spermatozoa. High and low ΔΨm in cooled stallion semen varied between stallions and between ejaculates, but was not affected by single-layer centrifugation (SLC). The SLC-selected spermatozoa produced significantly less hydrogen peroxide than controls (P < 0.001), which could explain the increased longevity and retention of fertilising capacity seen in previous studies. For SLC samples, ΔΨm was positively associated with viable spermatozoa that were not producing reactive oxygen species (r = 0.49; P < 0.001) and negatively associated with ROS production (for superoxide: r = -0.4, P < 0.01; for hydrogen peroxide: r = -0.39, P < 0.05). There was no clear association between ΔΨm and ROS production in control samples.

In vivo fertilizing ability of stallion spermatozoa processed by single layer centrifugation with Androcoll-E™

A colloid with a species specific silane-coated, silica-based formulation, optimized for stallion (Androcoll-E™), enables a better sub-population of spermatozoa to be selected from stallion ejaculates. However, such a practice has not been critically evaluated in stallions with fertility problems. In this study we evaluate whether single-layer centrifugation (SLC) through Androcoll-E™ could be used to enhance fertility rates in a subfertile stallion. Ejaculates were obtained from two different stallions, one Lusitano (fertile) and one Sorraia (subfertile), with distinct sperm characteristics and fertility. Motility, morphology, plasma membrane structural (eosin-nigrosin) and functional integrity (HOS test), mitochondrial functionality (Δψm; JC-1) and longevity (motility after 72 h cooling) after centrifugation in Androcoll-E™, as well as pregnancy rates obtained after artificial insemination (AI), with and without (control group) SLC-treated sperm were assessed. The effect of SLC on sperm characteristics, and fertility results were evaluated by ANOVA and Fisher procedures, respectively. Our results showed that SLC-selected sperm did not differ from the raw semen in terms of viability, morphology, response to hypo-osmotic conditions (HOS test) and mitochondrial membrane potential (↑ΔΨmit; JC-1). Sperm motility in cooled samples was not improved by SLC treatment. Our data show that SLC through Androcoll-E™ has no effect on pregnancy rates in the stallions used in this trial.

Effect of single-layer centrifugation or washing on frozen–thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?

The aims of this study were to determine the sperm quality of frozen–thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen–thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen–thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen–thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed.

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences (P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.

Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA

Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P &lt; 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA

Density gradient centrifugation with PureSperm® (PureSperm® 40+PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400×106spermmL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200×106spermmL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4cm above the surface of liquid nitrogen vapors for 10min, after which they were directly placed in liquid nitrogen. After 24 to 48h of storage, straws were thawed in a water bath at 37°C for 30s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P&amp;lt;0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65±0.05 v. 83.79±0.13; percentage of progressive motile spermatozoa: 79.38±6.66 v. 54.61±16.11), morphology (86.45±0.01 v. 83.51±0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32±0.04 v. 36.50±0.17; percentage of viable sperm with an acrosome reaction: 2.81±0.01 v. 9.74±0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

Single Layer Centrifugation of Stallion Spermatozoa through Androcoll™‐E does not Adversely Affect their Capacitation‐Like Status, as Measured by CTC Staining

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll™-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.

Comparison of density gradient and simple centrifugation of equine spermatozoa: effect on fertility of an oligospermic-subfertile stallion

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences (P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.