Short Hypoosmotic Swelling Test Research Articles

Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span.

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P < 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P < 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P < 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P < 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

Insemination of sows with seminal doses prepared by a two-step hypothermic dilution does not impair the reproductive performance at farm.

Reproduction in swine is mostly carried out through artificial insemination (AI). For this purpose, AI studs collect the ejaculates, analyse the sperm quality, dilute and package to produce seminal doses and ship them to sow farms to carry out the AI. Temperature is controlled during the process to avoid sperm damage. Semen is diluted in the extender in a one-step or a two-step process where the second can be isothermic (approximately 32°C) or hypothermic (room temperature 21-22°C). Both techniques are currently performed, and the latter could reduce time and costs, but the literature available comparing the processes is scarce and presents discrepancies. To date, there are no studies about its impact in fertility. This study compared hypothermic two-step dilution (HTSD) and isothermic two-step dilution (ITSD) in laboratory and field trial to elucidate whether HTSD has any effect. Ejaculates from 72 boars in nine AI studs were split and processed with both techniques using a high-performance extender and evaluated in laboratory. Four farms inseminated 345 sows with samples of four of these AI studs, and their fertility and prolificacy were registered. Results show no significant differences between doses prepared by HTSD and ITSD technique, having no impact in laboratory results (percentage of motile sperm, short hypoosmotic swelling test (sHOST) and short osmotic resistance test (sORT), viable sperm, damaged acrosomes, sperm under early apoptosis, high mitochondrial membrane potential (p>.1), fertility (92.2% versus 94.1%, p=.45) or farrowing rate (15.8±0.3 versus 16.1±0.3 p=.46). In conclusion, our results suggest that HTSD of semen on extender could be safely implemented in AI studs under the conditions tested.

Adverse effects of members of the Enterobacteriaceae family on boar sperm quality

Semen samples collected in 2012 from 1785 boars belonging to five different breeds were recruited from the quality control laboratory of Magapor SL, Spain. These samples came from 43 boar studs and resulted from diluting the ejaculates in commercial semen extenders. Evaluation of the semen sample characteristics (color, smell, pH, osmolality, concentration, motility of sperm cells, agglutination, acrosome integrity, short hypoosmotic swelling test, and abnormal forms) revealed that they met the international standards. The samples were also tested for the presence of aerobic bacterial contamination. In the present study, 14.73% (n = 263) of the semen samples were contaminated above 3 × 102 colony-forming units/mL with at least one type of bacteria. The Enterobacteriaceae family was by far the major contaminant, being present in 40.68% of the contaminated samples (n = 107). Bacterial strains of the Enterobacteriaceae family isolated from boar semen samples were in order of incidence (percentage of the contaminated samples): Serratia marcescens (12.55%), Klebsiella oxytoca (11.79%), Providencia stuartii (9.12%), Morganella morganii (3.80%), Proteus mirabilis (1.90%), and Escherichia coli (1.52%). We have seen that the presence in semen samples of S. marcescens, K. oxytoca, M. morganii, or P. mirabilis, but not P. stuartii or E. coli, was negatively associated with sperm motility (P < 0.05). The mean sperm concentration (P < 0.05), the mean percentage of spermatozoa with curled tails after the short hypoosmotic swelling test (P < 0.01), and the incidence of morphologically normal acrosomes (P < 0.05) were also lower in semen samples infected with M. morganii compared with uninfected ones. Moreover, P. mirabilis was negatively associated with the presence of abnormal forms. Thus, on the basis of the pathological effects that some of these strains may have on boar sperm quality, bacterial contamination should always be examined in semen samples prepared for artificial insemination.

Effects of long-term liquid commercial semen extender and storage time on the membrane quality of boar semen

The objective of this study was to assess the sperm membrane integrity in extended boar semen during storage time using specific spectrum laboratory methods. Boar semen was diluted with the long-term liquid commercial extenders Androhep (A), Androstar (AS), Androstar plus (AS&lt;SUP&gt;+&lt;/SUP&gt;), LD and M III and was stored up to 96 h. The sperm membrane integrity was evaluated by motility, viable spermatozoa, short hypoosmotic swelling test (sHOST) and by the activity of the enzyme aspartate aminotransferase (AST). Negative changes in the quality of sperm membrane in relation to storage time were observed after 48 h for sHOST, after 72 h for viable spermatozoa and after 72 h for motility. The percentage of viable spermatozoa was decreased by 0.27% each hour. A statistically significant difference between extenders A and LD was observed in sHOST after 72 h and 96 h storage (&lt;I&gt;P&lt;/I&gt; &amp;lt; 0.05). The AST activity did not show any statistically significant differences in extenders and in storage time. In overall assessment Androhep was the best of the tested extenders, followed by AS, AS&lt;SUP&gt;+&lt;/SUP&gt;, M III and LD in terms of motility, viable spermatozoa and sHOST. The correlations among laboratory methods were highly significant (&lt;I&gt;P&lt;/I&gt; &amp;lt; 0.001). In conclusion, the results documented that the sperm membrane integrity was statistically significantly affected by extenders and storage time (&lt;I&gt;P&lt;/I&gt; &amp;lt; 0.001). Boar semen quality was the best in extender A. sHOST is a very sensitive and relatively simple method for the assessment of sperm membrane integrity in diluted semen.

Effects of Straw Volume and Equex‐STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

Changes in subpopulations of boar sperm defined according to viability and plasma and acrosome membrane status observed during storage at 15 °C

Four boar ejaculates were preserved for 42 d at 15 °C to examine the changes produced in the quality of sperm membranes according to their response to a combined short hypoosmotic swelling test (sHOST) plus viability test designated the sHV test. Every 1 or 2 d, a sample from each ejaculate preserved in long-term extender was subjected to sperm motility determination and the sHV test. Through simultaneous examination by phase contrast and fluorescence microscopy, three subpopulations of sperm were identified according to their response to sHOST challenge and their viability status. In the subpopulations scoring positive in the sHOST, a further four sperm subpopulations were defined according to their viability and acrosome status. All the sperm subpopulations differed in terms of changes in their proportions produced during the course of preservation and individual differences among ejaculates were detected in terms of relationships shown among subpopulations. The combined sHOST/viability test was able to identify sperm subpopulations with the strongest plasma and acrosome membranes as well as a subpopulation of sperm that had undergone a true acrosome reaction.

Effects of exposure of pre-pubertal boars to di(2-ethylhexyl) phthalate on their frozen-thawed sperm viability post-puberty

Late effects of pre-pubertal oral exposure to di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in, for example, polyvinyl chloride-products, on semen quality in young boars have not been clear-cut. The aim of this study was to determine whether stress imposed on spermatozoa would reveal such effects. Semen was collected from post-pubertal boars (8-9 months of age), which had been exposed to 300 mg kg(-1) body weight of DEHP per os three times a week from 3 to 7 weeks of age and from control siblings given placebo (water). The semen was cryopreserved and examined for plasma membrane integrity post-thaw using the short hypo-osmotic swelling test and flow cytometry (propidium iodide /SYBR-14). Sperm motility was assessed by computer-assisted sperm analysis. No significant difference in plasma membrane integrity could be found between the groups. The DEHP-exposed group had a significantly lower percentage of linearly motile spermatozoa at 30 min (P < 0.05) and 120 min (P < 0.001) after thawing, and a larger amplitude of lateral displacement of the head 120 min after thawing (P < 0.05), compared with controls. In summary, spermatozoa from boars pre-pubertally exposed to low doses of DEHP, showed kinematic deviations post-thaw that could be related to DEHP exposure.

Four subpopulations of boar spermatozoa defined according to their response to the short hypoosmotic swelling test and acrosome status during incubation at 37 °C

This study was designed to confirm the previously observed relationship between response to the short hypoosmotic swelling test (sHOST) and acrosome resistance in boar spermatozoa. Ejaculates from 22 boars were incubated in a water bath at 37 °C for 2 h. During the incubation period, samples were taken at 5, 20, 40, 60, 90 and 120 min and subjected to the sHOST. sHOST responses (positive HP–negative HN) and acrosomal status (normal or intact NA–damaged DA) were evaluated in 100 spermatozoa corresponding to each ejaculate and incubation time, and the results used to establish four subpopulations: HPNA, HPDA, HNNA and HNDA. Over the entire incubation period, the sHOST positive subpopulation with damaged acrosomes, HPDA, was significantly smaller than the sHOST negative, damaged acrosome subpopulation, HNDA ( P<0.001). Further, proportions of HPDA spermatozoa remained stable throughout this period while the HNDA subpopulation showed a significant increase ( P<0.001) from the start to the end of incubation. These results confirm the high resistance of the plasma membrane of HP spermatozoa allowing the persistence of a higher number of intact acrosomes over time, compared to HN spermatozoa. Characterising this HPNA subpopulation may help the evaluation of ejaculate quality.

A short hypoosmotic swelling test for the prediction of boar sperm fertility

This study was designed to explore the relationship between the ejaculate response to a hypoosmotic swelling test (HOST) and in vivo fertility in a group of 38 boars. The hypoosmotic test used was a modification of the HOST that involves a shorter incubation time (5 vs 120 min) and lower osmotic pressure (75 vs 150 mOsm/kg). Ejaculates containing less than 20% abnormal spermatozoa were selected and checked for percentage of motility, percentage of normal acrosomes, percentage of short ORT and percentage of cells showing positive short HOST (sHOST) results. Two hundred eightyeight sows were inseminated to obtain in vivo fertility and prolificacy data. No differences were shown between technicians in the sHOST results obtained. Significant differences were recorded between boars in sHOST results (p < 0.002). Only the sHOST result presented a significant correlation with in vivo fertility (r = 0.43, p < 0.01). Short HOST data significantly improved fertility prediction of routine semen analysis tests. Unlike motility and acrosomal status, sHOST values showed a significant decrease when fresh ejaculates (37°C) were stored for 24 h at 15°C, indicating possible damage due to cold shock.