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Martínez García, Emilio

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Martínez García, Emilio
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Universidad de Murcia. Departamento de Medicina y Cirugía Animal
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Subject: Boar ×

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    Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?
    (Elsevier, 2018-08) Li, Junwei; Roca Aleu, Jorge; Pérez Patiño, Cristina; Barranco Cascales, Isabel; Martínez García, Emilio; Rodríguez Martínez, Heriberto; Parrilla Riera, Inmaculada; Medicina y Cirugía Animal
    This study aimed to elucidate the effect of seminal plasma (SP) from post-SRF on boar sperm freezability and, in addition, to determine the relevance of sperm itself to sustain cryopreservation, regardless of the SP surrounding them. Twelve ejaculates from three boars were manually collected in fractions/portions, P1: the first 10 mL of the SRF, P2: the rest of the SRF and the post-SRF. Immediately, samples were centrifuged to separate spermatozoa from the surrounding SP. Spermatozoa from P1 and P2 were then incubated with its own SP or that from post-SRF, diluted in BTS (1:1, v/v) at 17 °C overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P < 0.05) when P1- or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P < 0.05) regardless of the SP they were incubated in. Post-thaw sperm quality and functionality differed between P1 and P2, regardless of the SP used for overnight pre-freezing incubation. Post-thaw motility (P < 0.05) and viability (P < 0.01), as well as plasma membrane fluidity (P < 0.05) or lipid peroxidation values (P < 0.01) were best in P1 sperm compared to those of P2. The protein profile of sperm from P1 and P2, analyzed by 2D-PAGE, showed qualitative differences, which suggest that sperm rather than SP would explain differences in sperm freezability between ejaculate fractions/portions. Use of P1 fraction spermatozoa seems thus optimal for cryopreservation.
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    Seminal plasma antioxidants are directly involved in boar sperm cryotolerance
    (Elsevier, 2018-02) Li, Junwei; Barranco Cascales, Isabel; Tvarijonaviciute, Asta; Molina, Manuel F.; Martínez García, Emilio; Rodríguez Martínez, Heriberto; Parrilla Riera, Inmaculada; Roca Aleu, Jorge; Medicina y Cirugía Animal
    Boar ejaculates are ejected in fractions with a specific composition in terms of sperm numbers and seminal plasma (SP), which is reflected in the varying sperm cryotolerance observed among different fractions. As boar sperm are particularly sensitive to oxidative stress, this study evaluated the role of SP antioxidants in the observed differences in sperm cryotolerance among ejaculate fractions. Ten ejaculates from five boars were manually collected in fractions: the first 10 mL of the sperm-rich fraction (SRF), the rest of the SRF and the post-SRF. Semen samples comprising the entire ejaculate (EE) were created by proportionally mixing the three fractions described above. Each of the 40 resulting semen samples was split into two aliquots: one was used for sperm cryopreservation following a standard protocol utilizing 0.5-mL straws, and the other was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (the first 10 mL of the SRF and the rest of the SRF) and those from post-SRF were of the highest and worst quality, respectively, which was measured in terms of total and objective progressive motility and viability (P < 0.01). Viable FT sperm from the post-SRF generated more reactive oxygen species and experienced more lipid peroxidation than those from the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). The percentage of FT sperm exhibiting fragmented nuclear DNA did not differ among ejaculate fractions and the EE. Catalase, glutathione peroxidase and glutathione peroxidase 5 (GPx-5) were lowest in SP from the first 10 mL of the SRF (P < 0.001), whereas superoxide dismutase (SOD) and paraoxonase 1 (PON-1) were highest in SP of the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). Trolox-equivalent antioxidant capacity (TEAC) and the ferric-reducing ability of plasma (FRAP) were highest in SP from the first 10 mL of the SRF and lowest in the post-SRF (P < 0.001), whereas cupric-reducing antioxidant capacity was lowest (P < 0.05) in SP from the first 10 mL of the SRF. Regression analyses indicated that certain SP antioxidants had good predictive value for post-thaw recovery rates of total motility (R2 = 54.8%, P < 0.001; including SOD, TEAC and FRAP) and viability (R2 = 56.1%, P < 0.001; including SOD, PON-1, GPx-5 and TEAC). These results demonstrated that certain SP antioxidants are positively involved in boar sperm cryotolerance, minimizing the oxidative stress imposed by cryogenic handling.
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    Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa
    (Elsevier, 2013-08) Martínez Alborcia, María J.; Morrell, Jane M.; Gil Corbalán, María Antonia; Barranco Cascales, Isabel; Maside, Carolina; Alkmin, Diego V; Parrilla Riera, Inmaculada; Martínez García, Emilio; Roca Aleu, Jorge; Medicina y Cirugía Animal
    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.
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