Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculate.

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially post-thaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to mBO+ only induced minor variations. The data showed that kinetics, PMS and PMI of boar spermatozoa suspended in BTS (S1), LEY (S2) or LEY plus glycerol (S3) were maintained during controlled cooling but were altered by thawing, showing more characteristics of cell injury than of sperm capacitation. The spermatozoa were able to capacitate but the bicarbonate challenge destabilized the plasma membrane during initial cooling and accelerated membrane changes post-thaw. We conclude that capacitation of boar spermatozoa does not occur during controlled cooling.

Saravia F ，Hernández M ，Wallgren M ，Johannisson A ，Rodríguez-Martínez H ... -  《international journal of andrology》

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks.

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.

Saravia F ，Wallgren M ，Johannisson A ，Calvete JJ ，Sanz L ，Peña FJ ，Roca J ，Rodríguez-Martínez H ... -  《THERIOGENOLOGY》

Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation.

Boar spermatozoa collected in the ejaculate sperm peak-portion (P1, first 10 mL of the sperm-rich fraction, SRF), had shown a higher resilience to freezing and thawing compared to spermatozoa from the rest of the ejaculate (2nd portion of the SRF plus the post-sperm-rich fraction, PSRF), even when using a simplified freezing technique, as long as spermatozoa were incubated in their own seminal plasma (SP). This experiment studied the stability of P1- and SRF-P1 boar spermatozoa frozen in MiniFlatPacks (MFP), post-thaw, using flow cytometry. Since spermatozoa from either portion showed similar cryosurvival and low proportions of unstable membranes (<3%, annexin-V/propidium iodide staining), and only a tendency for SRF-P1 live spermatozoa to depict acrosome exocytosis (FITC-PNA/PI/H33342); they were explored for Ca(2+) contents using a Fluo-4 probe under in vitro capacitating conditions (mBO+ medium), as well they were tested for their ability to sustain a short Ca(2+)-ionophore (A23187) in vitro challenge. The proportions of live spermatozoa depicting high Ca(2+)-levels were initially <2% but increased over incubation time, particularly in SRF-P1(P<0.05), while proportions of live spermatozoa with low Ca(2+)-levels were basically constant over incubation time (~11-14%), for either portion. Incubation in capacitation medium did not modify the proportions of low-Ca(2+) but dramatically increased the proportions of high-Ca(2+) spermatozoa (P<0.001) already after 15 min exposure, highest for SRF-P1 spermatozoa. While the proportion of live spermatozoa with intact acrosome was significantly decreased among SRF-P1 (P<0.001), that of P1-spermatozoa remained unchanged, probably owing to the lowest relative content of cytosolic Ca(2+). The results suggest that spermatozoa in the P1-portion are more resilient to express acrosome exocytosis post-thaw compared to those bathing in the rest of the SRF-fraction when cryopreserved using a simplified technique, in MFPs.

Hossain MS ，Johannisson A ，Siqueira AP ，Wallgren M ，Rodriguez-Martinez H ... -  《-》

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen.

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).

Gil J ，Söderquist L ，Rodriguez-Martinez H 《THERIOGENOLOGY》

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 10mL (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 ([Ca(2+)]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 [Ca(2+)]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 32kDa 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.

Kumaresan A ，Siqueira AP ，Hossain MS ，Bergqvist AS ... -  《-》