Sperm-rich Ejaculate Fraction Research Articles

Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10–14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0–d5), T2 (d6–d10), and T3 (d11–d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Effects of age, size and season on cryotolerance of dog semen – A retrospective analysis

In a retrospective analysis of 508 ejaculates from 297 dogs, efficiency of semen cryopreservation and effects of age, season and dog size on characteristics of fresh and cryopreserved semen were evaluated. Volume of the sperm-rich ejaculate fraction increased to 10 years of age, decreased in 10 and 11-year old dogs (P < 0.001) but did not differ among seasons. Total sperm count was less in 10 and 11-year-old dogs (P < 0.001). The percentage of progressively motile, membrane-intact and morphologically normal spermatozoa before cryopreservation was least in 10 and 11-year-old dogs (P < 0.001). Cryopreservation resulted in less progressively motile spermatozoa (P < 0.001) with this being most pronounced in 10 and 11-year-old dogs (cryopreservation x age P = 0.004). The cryopreservation-induced decrease in morphologically normal spermatozoa (P < 0.001) was not affected by dog body weight. Number of cryopreserved AI doses differed among age groups (P < 0.001) and was less in 10 and 11-year-old dogs (median 1.5) compared with younger dogs (6–7 years, median 4.3). When ejaculates were grouped by a threshold of ≥ 35% progressively motile spermatozoa after freezing-thawing, 86.5% of all ejaculates were greater the threshold but this percentage decreased to 66% in 10 and 11-year-old dogs (P < 0.001). In conclusion, sperm cryotolerance is consistent for much of a dog´s lifespan but decreases after a certain age. Dog semen can be cryopreserved successfully throughout the year. Post-thaw semen characteristics were not different among cryopreserved ejaculates obtained from dogs differing in body weight.

The proteome of frozen-thawed pig spermatozoa is dependent on the ejaculate fraction source

The preservation of sperm functional parameters and fertility post-cryopreservation largely varies in the porcine, a species with a fractionated ejaculate. Although intrinsic individual differences have primarily been linked to this variation, differences in protein abundance among frozen-thawed (FT)-spermatozoa are far more relevant. This study, performed in two experiments, looked for proteomic quantitative differences between FT-sperm samples differing in post-thaw viability, motility, apoptosis, membrane lipid peroxidation and nuclear DNA fragmentation. The spermatozoa were either derived from the sperm-rich ejaculate fraction (SRF) or the entire ejaculate (Experiment 1) or from the first 10 mL of the SRF, the remaining SRF and the post-SRF (Experiment 2). Quantitative sperm proteomic differences were analysed using a LC-ESI-MS/MS-based SWATH approach. In Experiment 1, FT-spermatozoa from the SRF showed better preservation parameters than those from the entire ejaculate, with 26 Sus scrofa proteins with functional sperm relevance showing relative quantitative differences (FC ≥ 1.5) between sperm sources. In Experiment 2, FT-spermatozoa from the first 10 mL of the SRF and the remaining SRF were qualitatively better than those from the post-SRF, and 187 proteins showed relative quantitative differences among the three ejaculate sources. The results indicate that quantitative proteome differences are linked to sperm cryosurvival.

The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation.

Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.

Profile and reproductive roles of seminal plasma melatonin of boar ejaculates used in artificial insemination programs.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular HO generation, total and mitochondrial O production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons ( < 0.01) and day length periods ( < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75-21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02-35.61). The SP MLT also differed ( < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related ( < 0.001) to mitochondrial O production in viable sperm. The SP MLT did not differ among AI boars ( = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing1,2

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Characterization of the porcine seminal plasma proteome comparing ejaculate portions

Full identification of boar seminal plasma (SP) proteins remains challenging. This study aims to provide an extensive proteomic analysis of boar SP and to generate an accessible database of boar SP-proteome. A SP-pool (33entire ejaculates/11 boars; 3ejaculates/boar) was analyzed to characterize the boar SP-proteome. Twenty ejaculates (5 boars, 4ejaculates/boar) collected in portions (P1: first 10mL of sperm rich ejaculate fraction (SRF), P2: rest of SRF and P3: post-SRF) were analyzed to evaluate differentially expressed SP-proteins among portions. SP-samples were analyzed using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS followed by functional bioinformatics. The identified proteins were quantified from normalized LFQ intensity data. A total of 536 SP-proteins were identified, 409 of them in Sus scrofa taxonomy (374 validated with ≥99% confidence). Barely 20 of the identified SP-proteins were specifically implicated in reproductive processes, albeit other SP-proteins could be indirectly involved in functionality and fertility of boar spermatozoa. Thirty-four proteins (16 identified in S. scrofa taxonomy) were differentially expressed among ejaculate portions, 16 being over-expressed and 18 under-expressed in P1–P2 regarding to P3. This major proteome mapping of the boar SP provides a complex inventory of proteins with potential roles as sperm function- and fertility- biomarkers. Biological significanceThis proteomic study provides the major characterization of the boar SP-proteome with >250 proteins first reported. The boar SP-proteome is described so that a spectral library can be built for relative ‘label free’ protein quantification with SWATH approach. This proteomic profiling allows the creation of a publicly accessible database of the boar SP-proteome, as a first step for further understanding the role of SP-proteins in reproductive outcomes as well as for the identification of biomarkers for sperm quality and fertility.

High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility.

The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (n° = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 °C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (n° = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.

Effect of the holding time at 15 °C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15°C prior to cryopreservation (2, 4 and 8h), thawing rate (37°C for 20s or 70°C for 8s) and post-thaw incubation temperature (15°C or 37°C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240min of incubation. The results show that with the holding time at 15°C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70°C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37°C for 20s. In addition after thawing the sperm samples incubated at 15°C had a sustained sperm quality for longer, up to 4h post-thawing.

Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa

The goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H2DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.

Detrimental effects of non-functional spermatozoa on the freezability of functional spermatozoa from boar ejaculate.

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0 –native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H2DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa.

Micro disc gradient gel electrophoresis of seminal plasma proteins before and after vasectomy and in different split ejaculate fractions.

The seminal plasma proteins of patients before and after vasectomy as well as in different split ejaculate fractions were investigated using gradient polyacrylamide gel electrophoresis. Considerable interindividual variations of the protein patterns were observed. A distinct high molecular weight proteins band was found to disappear after vasectomy. This protein band could b4 attributed to the sperm-rich fraction of split ejaculates. It seems to be of testicular-epididymal origin and may be of interest for functional characterization of the testicular-epididymal fluid and for diagnosis of an occlusion of the excretory duct system.

75 GLUCOSE CONCENTRATION OF FREEZING EXTENDER MODULATES THE TYROSINE PHOSPHORYLATION PATTERN OF FROZEN-THAWED BOAR SPERMATOZOA

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance. Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.

Cryopreservation of Iberian red deer ( Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1 + 1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160 × 1 0 6 mL −1 and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl ® (20% egg yolk) and UL extender (Tes–Tris–fructose, 15% egg yolk, 4% glycerol). Triladyl ® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl ®, Andromed ®, Bioxcell ®, and UL with 8% LDL (low-density lipoproteins). Triladyl ® and Andromed ® performed better than Bioxcell ® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1 + 1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl ® and Andromed ® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.

Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks

In this study we demonstrate, in the frozen state, the architecture of frozen boar spermatozoa collected from the sperm-rich fraction of ejaculates ( n = 13) from four fertile boars packed and split-frozen in medium-straws (MS) and MiniFlatPacks (MFP), cross-sectioned in the frozen state and evaluated by image analysis on images obtained by use of cryo-scanning electron microscopy (Cryo-SEM). The tested hypothesis was that the degree of in situ dehydration and levels of homogeneity of boar semen either frozen in MSs or MFPs packages differ between them, with MFPs allowing for a more uniform dehydration of the spermatozoa and a higher cryosurvival, monitored by computer assisted sperm analysis (CASA) as proportion of linearly motile spermatozoa, compared to semen packaged and processed in MSs. The organization and relative surface of biological material (veins; e.g., frozen extender, bound water, solutes and spermatozoa) as well as free water (lakes) was measured as the degree of dehydration of the samples. The apparent organization of lakes and veins differed between packages, with the MFPs depicting larger lakes than the MSs. The sizes of the lakes in the latter appeared, moreover, highly asymmetrical depending on their position of the section. The relative surface of these lakes per section, respectively veins differed between packages ( P < 0.05), indicating a larger amount of free-water (lakes; 81.73 ± 2.07% vs. 77.91 ± 1.57%) in the MFPs and, consequently, thinner veins than in MSs. In conclusion, MFPs seem to allow for a more homogenous dehydration of the spermatozoa/frozen extender compared to MSs, which might account for their somewhat better sperm quality post-thaw.

Factors influencing boar sperm cryosurvival1

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.

130 EFFECTIVENESS OF BUTYLATED HYDROXYTOLUENE AS EGG YOLK SUBSTITUTE FOR CRYOPRESERVATION OF BOAR SPERMATOZOA

Butylated hydroxytoluene (BHT) has proven to be efficient as a supplement for cryopreservation boar spermatozoa (Roca et al. 2004 J. Androl. 25, 397–405). Moreover, it has been successfully used as an egg yolk substitute to cryopreserve goat spermatozoa (Khalifa and El-Saidy 2006 Anim. Reprod. Sci. 93, 303–315). The objective of this study was to evaluate the effectiveness of BHT as an egg yolk substitute for freezing boar spermatozoa. Nine sperm-rich ejaculate fractions were collected from 3 boars (3 ejaculates per boar) using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet of each ejaculate was split into 5 aliquots. The aliquots were diluted (to a final concentration of 1 � 109 sperm/mL) in a Tris-citric-glucose extender with 3% glycerol and supplemented with 20% egg yolk (positive control, PC aliquot) or BHT at the final concentrations of 0 (negative control, NC aliquot), 0.2, 0.4, and 0.8 mM. Diluted semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20�C min. Thawing was carried out in a water bath at 70�C for 8 s. Post-thaw sperm survival was assessed according to total sperm motility (TSM, %) using a CASA system (SCA�; Microptic, Barcelona, Spain), and plasma membrane integrity (PMI, %) and acrosome membrane integrity (AMI, %) using a flow cytometric procedure (SYBR-14/propidioum iodide/FITC-phycoerythrin), at 30 and 150 min post-thawing in diluted Beltsville thawing solution with spermatozoa held in a waterbath at 37�C (3 straws per ejaculate). Data were analyzed using a ANOVA mixed model including the main effects of aliquot, boar, post-thaw assessment time, and their interactions, with ejaculate and straw as random effects. All main effects had significant influence (P ≤ 0.01) in all post-thaw sperm assessments. However, no interactions (P ≥ 0.05) among main effects were shown. Data were combined for the 2 post-thaw assessment times. The best (P ≤ 0.05) post-thaw sperm quality (mean � SEM) was achieved in PC aliquots (47.11 � 3.10, 58.98 � 2.78, and 51.35 � 3.42 for TSM, PMI, and AMI, respectively). In NC aliquots, the percentage of TSM, PMI, and AMI were always below 1% (P ≤ 0.05). BHT has a beneficial (P ≤ 0.05) effect on post-thaw sperm assessments, and no differences (P ≥ 0.05) among concentrations were shown. The mean post-thaw sperm quality in the BHT aliquots was 8.50 � 0.80, 20.29 � 0.53, and 16.03 � 0.55 for TSM, PMI, and AMI, respectively. On the basis of these data, we can conclude that BHT has a protective effect for boar spermatozoa during the cryopreservation process. However, BHT alone is insufficient to replace the protective effect of egg yolk. This work was supported by CICYT (AGF2005-00706), Madrid, Spain.

Kinematic Changes During the Cryopreservation of Boar Spermatozoa

The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P > .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P > .05), sP2 and sP3 varied significantly (P < .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

Characterization and localization of membrane vesicles in ejaculate fractions from the ram, boar and stallion.

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.