Isabel Barranco

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 (500 g 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-H(2)DCFDA) 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.

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.

Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate.

Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.

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.

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008-2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24h at 17°C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P<0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the seasons influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.

The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility

Paraoxonase 1 (PON‐1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON‐1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP‐PON‐1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra‐boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP‐PON‐1 activity differed among ejaculate portions, showing the spermatozoa‐peak portion of spermatozoa‐rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post‐sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP‐PON‐1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP‐PON‐1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP‐PON‐1 activity differed among boars and ejaculate fractions/portions. SP‐PON‐1 activity was positively correlated with sperm quality and functionality of liquid‐stored semen samples and it evidenced a positive association with in vivo fertility.

Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates.

This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON‐1, extracellular) and type 2 (PON‐2, intracellular) in boar semen. To evaluate PON‐1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON‐1 and total cholesterol levels. Seminal‐plasma PON‐1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal‐plasma PON‐1 concentration and enzymatic activity were negatively correlated (r = −0.763; P < 0.01). The activity of seminal‐plasma PON‐1 negatively correlated with ejaculate volume (r = −0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal‐plasma cholesterol concentration positively correlated with PON‐1 activity (r = 0.773; P < 0.01), but negatively correlated with PON‐1 concentration (r = −0.709; P < 0.05). The presence of intracellular PON‐2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON‐2 localised to the post‐acrosomal area of the sperm head and principal piece of the tail in membrane‐intact spermatozoa. In summary, PON is present in boar semen, with PON‐1 at low levels in seminal plasma and PON‐2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal‐plasma parameters. Mol. Reprod. Dev. 82: 58–65, 2015.

The Seminal Plasma of the Boar is Rich in Cytokines, with Significant Individual and Intra‐Ejaculate Variation

The boar, as human, sequentially ejaculates sperm‐rich and sperm‐poor fractions. Seminal plasma (SP) spermadhesins (PSP‐I/PSP‐II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility.

Glutathione Peroxidase 5 Is Expressed by the Entire Pig Male Genital Tract and Once in the Seminal Plasma Contributes to Sperm Survival and In Vivo Fertility

Glutathione peroxidase-5 (GPX5) is an H2O2-scavenging enzyme identified in boar seminal plasma (SP). This study attempted to clarify its origin and role on sperm survival and fertility after artificial insemination (AI). GPX5 was expressed (Western blot and immunocytochemistry using a rabbit primary polyclonal antibody) in testes, epididymis and accessory sex glands (6 boars). SP-GPX5 concentration differed among boars (11 boars, P < 0.001), among ejaculates within boar (44 ejaculates, P < 0.001) and among portions within ejaculate (15 ejaculates). The first 10 mL of the sperm rich fraction (SRF, sperm-peak portion) had a significantly lower concentration (8.87 ± 0.78 ng/mL) than the rest of the SRF and the post-SRF (11.66 ± 0.79 and 12.37 ± 0.79 ng/mL, respectively, P < 0.005). Sperm motility of liquid-stored semen AI-doses (n = 44, at 15–17°C during 72h) declined faster in AI-doses with low concentrations of SP-GPX5 compared to those with high-levels. Boars (n = 11) with high SP-GPX5 showed higher farrowing rates and litter sizes than those with low SP-GPX5 (a total of 5,275 inseminated sows). In sum, GPX5 is widely expressed in the boar genital tract and its variable presence in SP shows a positive relationship with sperm quality and fertility outcomes of liquid-stored semen AI-doses.

Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled “Characterization of the porcine seminal plasma proteome comparing ejaculate portions” (Perez-Patiño et al., 2016) [1].