Browsing by Author "Lopez Sebastian, Antonio"

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Assessment of fertilizing ability of merino ram semen cold-stored up to 48h by heterologous in vitro fertilization of bovine oocytes
(CSIRO Publishing, 2019) Galarza Lucero, Diego Andrés; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Sánchez Calabuig, María Jesús; Lopez Sebastian, Antonio; Santiago Moreno, Julián; Rizos, Dimitrios; Galarza Lucero, Diego Andrés
The use of cold-stored ram semen has been applied in sheep artificial insemination programs, since it preserves its fertilizing ability similar to fresh. Besides, the heterologous in vitro fertilization (IVF) has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold-stored up to 48h at 5 ºC by assessing heterologous IVF using bovine oocytes. Fifteen pools of three normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200x106 spermatozoa/ml with UHT-based extender (skim milk-6% egg yolk) and cold-stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using either fresh semen (FS, n=707), cold-stored to 24h (CS24, n=832) or cold-stored to 48h (CS48, n=611). In parallel, homologous IVF (Control, n=1356) and parthenogenesis (Parth Control non-fertilized oocytes n=334) were performed. Ram non-selected and selected (BoviPure) semen parameters were evaluated by CASA. Sperm-oocyte interaction was assessed at 2.5 hours post-insemination (hpi) by evaluating the number of bound spermatozoa whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates was analysed using one-way ANOVA. Data was expressed as mean ± SEM. In terms of sperm storage time, non-selected semen showed a significant decrease (p<0.05) for CS24 and CS48h compared to FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5 vs 71.3±1.6] and straight-line velocity [VSL (mm/sec): 132.2±6.1 and 109.7±6.3 vs 176.7±4.3], respectively. However, selected semen showed a decrease (p<0.05) only for CS48h when compared to CS24h or FS on SPM (35.6±3.9 vs 56.1±6.91 and 59.3±2.6) and VSL (83.5±4.4 vs 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared to heterologous FS group (44.4±6.8 vs 12.5±4.5%, p<0.01). The polyspermy was higher in heterologous CS24 group when compared to homologous IVF (11.4±3.4 vs 3.8±2.2, p<0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi than heterologous IVF with FS (67.3±5.8 vs 35.2±5.6%), CS24 (72.1±4.5 vs 37.2±5.7%) and CS48 (63.0±6.0 vs 27.0±5.6%), respectively (p<0.001). Likewise, cleavage rate was higher in homologous group compared to heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8 vs 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 vs 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 vs 43.3±3.5 and 4.3±1.2%), respectively (p<0.001). In conclusion, Merino ram semen cold-stored up to 48h maintains its fertilization ability in the same extend as fresh and can be used for sheep crossbreeding programs
Effect of L-carnitine in a skimmed milk based-diluent on membranes preservation and kinetic activity of ram sperm under chilled conditions
(Asociación Interprofesional para el Desarrollo Agrario, 2019) Galarza Lucero, Diego Andrés; Lopez Sebastian, Antonio; Santiago Moreno, Julián; Galarza Lucero, Diego Andrés
The synergic effect of L-carnitine (LC) and skimmed milk base-extender (UHT) on the kinetic parameters and membranes integrity of cold-stored ram sperm was evaluated. For this purpose, twelve pools of thirty-six semen ejaculates that were collected with artificial vagina from twelve Merino rams were used. After initial evaluation, the semen was divided into 6 aliquots enough to diluted with UHT-extender (control) and 1mM (UHT-LC1), 2.5mM (UHT-LC2.5), 5mM (UHT-LC5), 7,5mM (UHT-LC7.5) and 10mM (UHT-LC10) of LC, respectively, at 200 x 106 cells/ml concentration. Semen samples were cold-stored (5ºC) up to 96 h. Kinetic variables and membranes integrity were assessed by CASA system (SCA) and triple fluorescence association test (IP/PNA-FITC/Mitotracker green). An ANOVA-one way and Bonferroni´s test were used to evaluated effects of LC doses. Overall, the total motility (TM, %), straight line velocity (VSL, μm/s) and total sperm with plasma and acrosome membranes integrity and high mitochondrial function (IPIAHM, %) were greater with all doses of LC (1 to 10 mM) than control group. Also, the UHT-LC5 group provided better values than others LC groups according to motilities. In conclusion, LC improves kinetic vigor and provides an antioxidant protective effect to sperm membranes of cold-stored ram when added to skimmed milk based-diluent
Influence of sperm filtration and the addition of glycerol to UHT skimmed milk- and TEST-based extenders on the quality and fertilizing capacity of chilled ram sperm
(2019) Galarza Lucero, Diego Andrés; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Sánchez Calabuig, María Jesús; Rizos, Dimitrios; Lopez Sebastian, Antonio; Santiago Moreno, Julián
The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, mm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with nonfiltered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.
Sephadex filtration as successful alternative to density-gradient centrifugation procedures for ram sperm selection with improved kinetics
(2018) Galarza Lucero, Diego Andrés; Lopez Sebastian, Antonio; Woelders, Henry; Blesbois, Elisabeth; Santiago Moreno, Julián
Density-gradients centrifugation (DGC) and filtration columns (FC) are used to separate deformed or dead sperm, debris, and other cells that may negatively affect the fertilizing capacity of sperm in fresh, chilled and frozen/thawed semen. The present study was conducted to evaluate the suitability of DGC (BoviPure®, Percoll® and Accudenz®) and FC (Sephadex G-15®) sperm selection procedures for fresh-extended and cold-stored ram semen by assessment of post-treatment sperm quality variables. Twenty normospermic ejaculates from ten adult Merino rams were used. Sperm concentration of recovered cells was greater (P < 0.001) after BoviPure treatment than other procedures in both fresh and cold semen. With the Sephadex method, there were more desirable values than with use of DGC procedures in several sperm motility variables measured by using the CASA system. In non-refrigerated semen samples, the percentage of progressive sperm motility (%PSM) after Sephadex filtration was greater (P < 0.05) than after BoviPure treatment; the straightline velocity (VSL) value after Sephadex filtration was greater (P < 0.01) than after Accudenz treatment; the amplitude of lateral head displacement (ALH) after Sephadex and Accudenz treatment was less than non-filtered semen (P < 0.001) and after Percoll (P < 0.01) and BoviPure (P < 0.05) treatments. In cold-stored semen samples, the %PSM after Sephadex filtration was greater than non-filtered (P < 0.05) semen and after BoviPure (P < 0.05), Percoll (P < 0.05) and Accudenz (P < 0.001) treatments. It is concluded that Sephadex column filtration can be used to select ram sperm in non-refrigerated and cooled semen, because percentage progressively motile sperm and some other sperm motility characteristics are greater with use of this techniques as compared with use of DGC methods.
Sperm selection by density-gradient centrifugation of merino ram semen cold-stored up to 48 h improves viability and membrane integrity
(2018) Galarza Lucero, Diego Andrés; Rizos, Dimitrios; Lopez Sebastian, Antonio; Santiago Moreno, Julián; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Galarza Lucero, Diego Andrés
Liquid ram semen stored at 5ºC would be more competent than frozen/thawed for sheep crossbreeding programs. The aim was to evaluate the kinetics and membrane integrity of Merino ram semen cold-stored up to 48h at 5ºC before and after density-gradient centrifugation (DGC) selection. Pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected by artificial vagina in fifteen sessions (45 ejaculates), diluted to 200x106 spermatozoa/ml with skim milk-based extender contained 6% egg yolk and cold-stored up to 48h at 5ºC. Motile spermatozoa were separated by BoviPure DGC (Galarza et al., 2018, Anim Reprod Sci 192: 261-270) using 250ul of fresh (n = 30) and cold-stored semen (24h: n = 10 and 48h: n = 10). The final pellet of 300ul was used to assess semen quality. The kinetic parameters were evaluated by computer-assisted sperm analysis (CASA) while plasma, acrosomal and mitochondrial membrane status was analyzed by PI/FITC PNA/Mitotracker fluorescence. The effects of storage time (fresh, 24 & 48h) and sperm selection process were analysed by univariant ANOVA and Bonferroni´s test (P < 0.05). In terms of sperm storage time, CASA analysis of non-selected semen samples showed a significant decrease after storage for 24 and 48h compared to fresh samples with regard to progressive motility [SPM (%): 52.30 ± 4.1 and 36.9 ± 5.5 vs 71.3 ± 1.6], straight line velocity [VSL (um/sec): VSL 132.2 ± 6.1 and 109.7 ± 6.3 vs 176.7 ± 4.3], linearity [LIN (%): 69.2 ± 3.5 and 59.0 ± 5.0 vs 82.0 ± 1.2], and straightness [STR (%): 75.7 ± 3.3 and 66.0 ± 4.3 vs 86.9 ± 0.9], respectively. However, analysis of DGC-selected semen showed a decrease only at storage for 48h when compared to 24h or fresh samples with regards to SPM (35.6 ± 3.9 vs 56.1 ± 6.91 and 59.3 ± 2.6), VSL (83.5 ± 4.4 vs 105.3 ± 6.5 and 110 ± 2.0) and LIN (63.9 ± 3.4 vs 75.0 ± 3.7 and 80.7 ± 2.4), respectively. A comparison between DGCselected and non-selected samples showed a significant lower total motility [TM (%): 94.4 ± 0.8 vs 85.4 ± 1.90], VSL (176.7 ± 4.2 vs 110.0 ± 2.0) and wobble [WOB (%): 94.2 ± 0.6 vs 88.5 ± 1.5] only for fresh semen. Fluorescence analysis evidenced a decrease only in 24h cold-stored non-selected compared with fresh semen with regard to plasma membrane integrity [PMI (%): 64.8 ± 2.9 vs 80.1 ± 1.7], high mitochondrial function [HMF (%): 88.2 ± 1.6 vs 93.9 ± 1.0] and total intact plasma/intact acrosome/high mitochondrial function [IPIAHM (%): 61.8 ± 3.1vs 78.7 ± 2.0]. In contrast, no differences were observed between fresh and cold-stored DGC-selected semen. A comparison between selected and non-selected semen showed a significant increase of PMI (64.8 ± 3.14 to 89.4 ± 2.32), HMF (88.2 ± 1.26 to 96.0 ± 1.26) and IPIAHM (61.8 ± 3.14 to 87.6 ± 2.04) only for 24h. These results suggest that kinetic activity of cold-stored and DGC-selected ram spermatozoa is maintained and the selection process results in improved viability and membrane integrity. Therefore, liquid storage combined with DGC selection might become a good alternative to fresh or frozen non-selected semen to be used for artificial insemination in sheep crossbreeding programs.