I Casas

Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels.

The main aim of the present study was to determine whether differences in the amounts of free cysteine residues in sperm nucleoproteins, which are a direct marker of the integrity of the disulfide bonds between nucleoproteins, existed between good (GFE) and poor boar freezability ejaculates (PFE) during the different steps of the freeze-thawing process. The analyzed steps were: (1) immediately before starting cryopreservation (17 °C), (2) at the end of the cooling step (5 °C), and (3) 30, and (4) 240 minutes after thawing. In addition, the present study also sought to determine whether GFE and PFE differed in the amounts of peroxides and superoxides generated during freeze-thawing as an overall measure of the boar sperm reactive oxygen species (ROS) accumulation rate. According to our results, PFE present lower resistance than GFE to cryopreservation-induced alterations of disulfide bonds between nucleoproteins, because levels of cysteine free residues were higher in PFE than in GFE at 30 and 240 minutes after thawing. On the other hand, no significant differences were observed between GFE and PFE in ROS levels during freeze-thawing. In conclusion, PFE are less resistant than GFE to cryopreservation not only in terms of sperm motility and membrane integrity, but also in the integrity of nucleoprotein structure. However, this difference between PFE and GFE in the resistance of the nucleoprotein structure to freeze-thawing is not linked with concomitant changes in ROS levels.

The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability.

In a previous study we reported that the immunolabelling of GLUT3, HSP90AA1, and Cu/ZnSOD proteins on boar sperm did not show differences between good and poor freezability ejaculates, in terms of a qualitative analysis based on location and reactivity of these proteins at 17 degrees C and at 240 min post-thaw. Since predicting the ejaculate freezability is considerably important in sperm cryopreservation procedures, the objective of the present study was to quantify the expression of these three proteins in good and poor freezability ejaculates. For this purpose, 10 ejaculates from 9 Piétrain boars were cryopreserved and their sperm quality assessed in the three main steps of the freezing process (17 degrees C, 5 degrees C, and 240 min post-thaw). After this assessment, the 10 ejaculates were clustered for freezability on the basis of their sperm progressive motility and membrane integrity at 240 min post-thaw. From the whole ejaculates, only four good and four poor freezability ejaculates displaying the most divergent values were selected for a western blot assay using sperm samples coming from the three mentioned freezing steps. Protein levels through densitometry were significantly different between good and poor freezability ejaculates for Cu/ZnSOD at 240 min post-thaw (P <or= 0.01) and for HSP90AA1 at 17 degrees C and 5 degrees C (P <or= 0.05). This last finding claims the introduction of tests based on molecular markers in spermatozoa to accurately predict the freezability of ejaculates in order to promote the use of frozen semen on artificial insemination programmes.

Effects of filtration of semen doses from subfertile boars through neuter Sephadex columns.

This study was designed to develop a method of improving the quality of sperm obtained from subfertile Piétrain boars. Seminal doses were filtered through neuter Sephadex columns (G-25 Medium, G-50 Fine, G-50 Medium and G-75, length 10 +/- 0.5 cm, flow rate 1 ml/20 s). Doses were prepared by pooling 10 ml semen samples collected from 58 asthenoteratospermic boars and diluted the sperm-cell rich fraction 1 : 6 in Betsville thawing solution extender. Sperm quality was determined before and after the filtering process. Sperm morphology and motility were assessed using the computer program SCA 2002 production, and sperm vitality was evaluated by fluorescence multistaining. ORT and HRT tests were used to determine the osmotic resistance of spermatozoa, and metabolic performance was assessed by measuring l-lactate production. Results indicate that the filtration process rendered increased proportions of mature spermatozoa and of viable spermatozoa with an intact acrosome, nucleus and mitochondrial sheath. Sperm filtration led to decreased percentages of spermatozoa with proximal and distal droplets and of agglutinated spermatozoa, along with slightly diminished ORT values. HRT scores and L-lactate production were unaffected. Our findings indicate that filtering through a Sephadex column improves the sperm morphology and vitality of seminal doses obtained from subfertile boars, but produces no functional changes in the spermatozoa. All four column types yielded similar results.

Relationship of sperm small heat-shock protein 10 and voltage-dependent anion channel 2 with semen freezability in boars

Freezability differences between boar ejaculates exist, but there is no useful method to predict the ejaculate freezability before sperm cryopreservation takes place. In this context, the present study sought to determine whether the amounts of small heat-shock protein 10 (also known as outer dense fiber protein 1) (ODF1/HSPB10) and voltage-dependent anion channel 2 (VDAC2) may be used as boar sperm freezability markers. With this aim, 26 boar ejaculates were split into two fractions: one for protein extraction and the other for cryopreservation purposes. Ejaculates were subsequently classified into two groups (good freezability ejaculates [GFE] and poor freezability ejaculates [PFE]) based on viability and sperm motility assessments after 30 and 240 minutes of after thawing. Although the VDAC2 amounts, analyzed through Western blot, were significantly higher (P < 0.01) in GFE (1.15 ± 0.18 density mm(2)) than in PFE (0.16 ± 0.03 density mm(2)), no significant differences were observed in ODF1/HSPB10 between both groups (i.e., 1.97 ± 0.38 density mm(2) in GFE vs. 1.87 ± 1.54 density mm(2) in PFE). In addition, principal component and multiple regression analyses indicated that the component explaining most of the variance (78.41%) in ejaculate freezability at 240 minutes after thawing resulted to be significantly (P < 0.05) correlated with VDAC2 content. This result revealed that the amounts of VDAC2 but not those of ODF1/HSPB10 may be used to predict the freezability of a given boar ejaculate before starting cryopreservation procedures.

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.

The protective effect of a 17 °C holding time on boar sperm plasma membrane fluidity after exposure to 5 °C

The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17°C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5°C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5°C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0h at 17°C; T2) Fresh diluted semen, 24h at 17°C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3h at 5°C; T4) Sperm from T2 in LEY, 3h at 5°C; T5) Sperm from T1 in LEY, 24h at 5°C; T6) Sperm from T2 in LEY, 24h at 5°C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane.

Boar sperm thawing practices: the number of straws does matter.

The number of straws thawed has been largely neglected in reports of boar sperm cryopreservation. Whereas previous studies confirm the effect of sperm concentration on function and survival of thawed boar spermatozoa, it is still unknown whether, for a same concentration, total number of sperm in the thawing solution affects its mechanics. The present trial sought to define good boar sperm thawing practices by checking if a minimal number of straws as well as the percentage of air volume in the thawing tube should be stated or not to decrease variability from one trial to another. In a first assay, three tubes with different numbers of thawed straws were compared in terms of motility and membrane integrity: control (C, four straws), T1.1 (two straws), and T1.2 (one straw). In a second parallel assay, the sperm motility was evaluated when one straw was thawed in a tube containing 86.67% of air volume (T2.1), and when the tube contained < 1% air volume (T2.2). In all treatments the final concentration of sperm in Beltsville thawing solution (BTS) was 1:3 (v:v) and quality parameters were assessed 4 h after thawing. Results showed the number of straws does affect motility parameters but not the membrane integrity, whereas less air volume in the tube nonsignificantly minimizes data deviation among replicates. In conclusion, it is recommended the use of four straws at 1:3 (v:v) to maintain motility records in boar sperm thawing practices as well as to be provided with vials that fit the sperm volume.

Gene Banking: The Freezing Strategy

Genetic resource banking (GRB) or ex situ conservation in livestock species is the storage of genetic material for breeding purposes. Genes are present, with few exceptions, in every cell within an organism but only germ cells have the ability to transmit this information from one individual to another. Germplasm banking is the main tool for directing and enhancing this genetic flow in intensive farming, as it is for preserving the genetic diversity of a livestock population. The only current technique for the storage of germ cells is freezing them in liquid nitrogen (cryobanking). Traits in the breeding goals of a genetic program are retained in this way from the moment of insemination; otherwise, these genetic resources would be lost. Sperm from boars displaying high genetic values can be preserved following either rapid or slow freezing, as it is explained in this chapter together with a complete introduction to the legal issues of animal cryobanking. A new way of sperm preservation in pigs is also discussed.

Effects of matrix filtration of low-quality boar semen doses on sperm quality.

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 +/- 0.5 cm), CM Sephadex (length 5 +/- 0.5 cm), glass wool (length 2 +/- 0.5 cm) or glass bead (length 10 +/- 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca((R)) 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l-lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l-lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.

Testing an egg yolk supplemented diet on boars to aid in sperm adaptation at 5°C.

Abstract In many species, extended semen can be stored at low temperatures to slow bacterial growth. However, boar semen performs poorly at temperatures below 15°C and this poses unique challenges, as it is not easy to maintain a constant 15–19°C during shipment. Some extenders have been formulated with egg yolk for storage at 5°C but the addition of egg yolk is not applicable in the majority of commercial operations. The purpose of this study was to evaluate if boar dietary supplementation with powdered egg yolk imparts any protective effects on sperm quality when stored at 15°C and 5°C for up to 11 days in a conventional extender. Ten boars were fed a commercial diet with the addition of 0.11Kg of powdered egg yolk for 10 weeks. Ejaculates collected on weeks 4, 6, 8, and 10 were processed for storage at both 15°C and 5°C and compared with ejaculates from boars fed a standard diet. Throughout an 11-day storage period, sperm quality was assessed including several motility and morphologic parameters and select plasma membrane properties (fluidity, integrity, and triacylglycerol content). Linear regression models were used to describe effects of treatment, storage day, week and temperature on all sperm parameters. Overall, there were minimal beneficial effects of egg yolk treatment on sperm quality parameters. Sperm from egg yolk supplemented boars did have a slower decline in viability and plasma membrane fluidity than that observed in the control sperm when stored at 5°C (p < 0.001). Additionally, there was an increase in total morphologic abnormalities in sperm from egg yolk fed boars compared to controls at week 10 (p < 0.001). In conclusion, the results of this study do not support a significant benefit to sperm quality or resistance to cold storage when feeding a 10-week dietary supplementation of 0.11Kg powdered egg yolk to crossbred boars.