Boar sperm changes after sorting and encapsulation in barium alginate membranes

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.     

Chlortetracycline analysis of boar spermatozoa after incubation,flow cytometric sorting,cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 μg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38°C, 4 hr), flow sorting, cooling (to 15 or 5°C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5°C (30.4, 48.5, 21.1%) than in those cooled to 15°C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively). Mol. Reprod. Dev. 46:408–418, 1997. © 1997 Wiley-Liss, Inc.     

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after “in vitro” capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.     

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.     

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 x g/5 min) just after sorting and stored 16-18 h at 17 degrees C (groups Cc and Hc) at 6 x 10(6)sperm/mL. The second aliquot was directly stored at 17 degrees C for 16-18 degrees C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 x 10(6)sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 x 10(6) sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16-18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower (p<0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer (p<0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.     

The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C

Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 μM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 μM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Assessment of sperm viability,mitochondrial activity,capacitation and acrosome intactness in extended boar semen during long-term storage

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.     

Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis

Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender.     

Single layer colloid centrifugation technique improves motility,viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.     

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.     

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.     

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.     

Assessment of acrosome-reacted boar spermatozoa using monoclonal antibody GB 24 and propidium iodide

Fluorescein-labeled GB 24, a mouse monoclonal antibody, was evaluated as an acrosomal dye for boar spermatozoa that had previously been stained with propidium iodide (PI) to assess sperm viability. A specific sperm-staining pattern with fluorescein-labeled GB 24 was shown to be associated with acrosome reaction on freshly ejaculated sperm when fixed with acetone or induced with ionophore A 23187, whereas the presence of PI staining was typical of dying spermatozoa. The GB 24-PI procedure was as accurate as the glutaraldehyde method in assessing acrosomal presence or absence on freshly ejaculated spermatozoa when spontaneous or A 23187-induced acrosomal reactions were considered. Approximately half of A 23187-induced spermatozoa with acrosomal loss did not exhibit a PI fluorescence; these were potentially viable acrosome-reacted spermatozoa. On semen diluted in a boar sperm-specific diluent (BTS-A) and stored, percentages of spermatozoa with nonintact acrosome from glutaraldehyde and GB 24-PI were not significantly different. Conversely, data from GB 24-PI was significantly lower than those from glutaraldehyde when semen were undiluted. This suggested that spermatozoa with reacted acrosome gradually lost their ability to bind with GB 24. Providing unequivocal and rapid scoring of acrosome-reacted spermatozoa, the GB 24-PI procedure may be a valuable tool in the evaluation of the acrosomal status of porcine fresh spermatozoa.