Subjecting canine semen to the hypo-osmotic test

doi:10.1016/0093-691X(93)90230-3

Theriogenology

Volume 39, Issue 6, June 1993, Pages 1279-1289

https://doi.org/10.1016/0093-691X(93)90230-3 Get rights and content

Abstract

The hypo-osmotic swelling test (HOS-test) was evaluated in 8 postpubertal dogs, ranging in age from 10 months to 3 years, to determine the response of canine spermatozoa to the HOS-test and the association between the sperm response to the test and semen parameters as well as to assess the effects of heating and storing semen on the response of the spermatozoa to the HOS-test. As in the human and bovine species, the canine spermatozoa curled (‘swelled’) under hypo-osmotic conditions, due to influx of water and expansion of the sperm membrane. Both fructose and sodium citrate solutions induced easily identifiable curling of spermatozoa, but with significant variations in sperm response to the 2 solutions. A maximal number of curled spermatozoa was observed in 60 mosmol fructose, at 45 minutes of incubation. Using the 60 mosmol fructose solution, a high correlation (r=0.94 P<0.0001) was observed between the percentage of curled spermatozoa and sperm motility. This is not surprising since sperm motility is partly dependent on membrane transport (membrane integrity). Comparatively, the correlations between curling and midpiece abnormality (r=0.22 P>0.32), and between curling and tail abnormality (4=0.38 P>0.08) were low. Storage of semen adversely influenced the sperm response to the HOS-test. The results of this study indicate the possible predictive value of the HOS-test in selecting individual ejaculates from stored canine semen samples for insemination. In conclusion, the HOS-test may be a useful addition to standard semen analysis for the identification of males that may be subfertile despite a normal spermiogram. Further evaluation of the test is needed to assess its diagnostic significance in this species.

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Canine Semen Evaluation and Processing
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Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?
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Citation Excerpt :
Results from the present study seem to corroborate the results from this previous study because there were different extents of curving/twisting in sperm incubated in hypo-osmotic conditions when EY was not included in the diluent which may indicate there was a different response of spermatozoa at the structural level. The capacity of dog spermatozoa for modification of the tail morphology in hypo-osmotic conditions was reported by Kumi-Diaka (1993). Unfortunately, in this previous study the extent of the sperm response to the hypo-osmotic stress was not reported; thus, it was not possible to compare the data from this previous and the present study directly.
Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).
Prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen
2016, Animal Reproduction Science
Citation Excerpt :
Following this process, the samples were immediately inserted into pre-cooled, labeled plastic straws (4 °C), which were sealed with a metal ball and stored at 4 °C for 20 min. For cryopreservation, the straws were placed horizontally in an isothermal Styrofoam box for 20 min, positioned 4 to 5 cm above the liquid nitrogen level before the samples were plunged into liquid nitrogen for storage for 7 days (Kumi-Diaka, 1993). After a 7-day period in the frozen state (England, 1993), semen was thawed according to the manufacturer’s instructions MOFA—Minitube of America (Verona, WI, USA).
Throughout cryopreservation, sperm are exposed to major osmotic challenges. Only intact membranes of sperm cells are able to regulate these volumetric changes, which can be determined by the hypo-osmotic swelling test (HOS test). Correlations between the HOS test and conventional semen variables are inconsistent. Therefore, the objectives of this study were (1) to examine relationships between HOS test results and standard semen variables before freezing and after thawing and (2) to evaluate the prognostic value of the HOS assessments on post-thaw quality of dog semen. Semen of 35 dogs was collected and analyzed before freezing and after thawing following a 7-day freeze–thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity morphology were evaluated and the HOS test was conducted with results from this test being recorded. In fresh semen the HOS test was positively correlated with progressive motility of sperm cells: r = 0.52, sperm cell membrane integrity: r = 0.50 and normal sperm cell morphology: r = 0.46 (P < 0.05). In frozen–thawed semen, the data obtained with the HOS test were positively correlated with progressive sperm cell motility: r = 0.67 and membrane integrity: r = 0.86 (P < 0.05). The data obtained with the HOS test in fresh semen were positively correlated with sperm cell membrane integrity: r = 0.50 normal sperm cell morphology: r = 0.55 and data from the HOS test (r = 0.43; P < 0.05) with frozen–thawed semen. For the prediction of individual cryopreservation capacity, results from assessment of the fresh semen variables of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen–thawed dog semen using the HOS test.
A commercial box for dog semen transport: What happens inside when the environmental temperature is increasing?
2014, Animal Reproduction Science
Citation Excerpt :
Observations in the present study enlighten aspects on storage temperatures for chilled dog semen and indicate the need for further studies to assess the impacts of addition of antibiotics on sperm stability and effectiveness at greater temperatures (above 15 °C) for more efficient ROS scavenger mechanisms. The resistance of sperm to osmotic stress is a functional sperm variable that has been positively correlated with sperm quality in dogs (Kumi-Diaka, 1993; Rodriguez-Gil et al., 1994) and with fertility in other species (Men: Check et al., 1988; Boar: Petrunkina et al., 2004; Bull: Khalil et al., 2006). For testing this feature in the present study, a flow cytometric approach was used which evaluated the integrity of the plasma membrane and the acrosomal membrane after incubation in isotonic and hypotonic media at 38 °C.
Environmental temperatures may influence the temperature inside commercial transport boxes during semen shipment and thereby storage conditions of diluted dog semen. To evaluate the temperature changes inside boxes and their influence on sperm quality, split semen samples (n = 8) were placed in Neopor boxes^® exposed for 48 h to room temperature (RT) (Box 1), 40 °C for 6 h and then kept at RT (Box 2) or 40 °C (Box 3). A fourth subsample was kept at 4–5 °C in a refrigerator (control). Inside Box 1 temperature initially decreased to <3 °C before it stabilized at 7–8 °C, while in Box 2 no decrease occurred and temperature was at 7–8 °C for 48 h. Temperature inside Box 3 was at 14–15 °C for 24 h and, thereafter, increased to 36.1 °C. Analysis of sperm motility (CASA) and viability (PI and FITC-PNA) after 24 and 48 h revealed marked sensitivity of dog spermatozoa to temperature fluctuations (Box 1). A constant storage temperature of 7–8 °C (Box 2) provided the most desirable semen quality in terms of motility, viability, as well as osmotic resistance when samples were stored for 48 h. Furthermore, results indicate that during 24 h preservation a storage temperature of 14–16 °C may provide optimum conditions for maintenance of sperm viability and function. An increase of the inside temperature to >30 °C (Box 3) resulted in an almost complete loss in sperm integrity. In conclusion, results suggest a revision of current recommendations for storage temperature of diluted dog semen. Boxes for semen transport should be prepared depending on the expected environmental temperatures.
Evaluation of testicular artery blood flow by Doppler ultrasonography as a predictor of spermatogenesis in the dog
2013, Research in Veterinary Science
Citation Excerpt :
Semen quality is traditionally measured by some physical parameters such as volume, sperm concentration, sperm motility, vitality and spermatozoa morphology (Amann, 1981). Recently, the hypo-osmotic swelling test (HOST) for assessing the integrity of the sperm membrane (Spittaler and Tyler, 1985) has been applied for evaluation of semen quality in man (Jeyendran et al., 1984; Fazano et al., 1993; Oosterhuis et al., 1996), bull (Correa and Zavos, 1994), boar (Lechniak et al., 2002) stallion (Neild et al., 1999; Nie and Wenzel, 2001), and dog (England and Plummer, 1993; Kumi-Diaka, 1993; Goericke-Pesch and Failing, 2012). The hypo-osmotic swelling test involves submersion of spermatozoa into a hypo-osmotic medium.
The present study examines the relationship between reproductive and seminal characteristic with blood flow supply of the testis in male dogs by means of Doppler ultrasonography. Five male dogs were subject to a clinical examination, blood sampling, B-Mode and Doppler ultrasonography of the testis, semen evaluation at collections and after storage at 4 °C for 24, 48 and 72 h in three replicates 30 days apart. Peak Systolic Velocity was positively correlated with testicular volume and negatively with live sperm at collections and after 24 h. A negative correlation existed between Resistive Index and Pulsatility Index with total and progressive motility at collections and also after 24, 48 and 72 h. Resistive Index and Pulsatility Index were also negatively correlated with the percentage of membrane intact sperms with curled tails at collections while the latter was positively correlated with End Diastolic Velocity. Moreover, positive linear correlations of weight with bilateral testicular volume and total sperm concentration and also between age and abnormal sperm at collections and after 24 h were established. The results suggest that the Resistive Index and Pulsatility Index seem to be potential markers of seminal quality in dogs.
Effect of l-Arginine and spermine-NONOate on motility, viability, membrane integrity and lipid peroxidation of Murrah buffalo (Bubalus bubalis) spermatozoa
2013, Livestock Science
Citation Excerpt :
In our study freshly ejaculated Murrah bull spermatozoa showed morphologic changes, as evidenced by curling–swelling, when exposed to hypo-osmotic milieu. And the response of buffalo bull semen to HOSt condition was similar to that observed in dog (Kumi-Diaka, 1993), human (Jeyendran et al., 1984), goat (Nur et al., 2005), bull (Correa et al., 1997; Nur et al., 2003), boar (Vazquez et al., 1997) and stallion (Neild et al., 1999). Moreover, spermatozoa extended and cryopreserved either in presence of spermone-NONOate or l-Arginine showed more tail coiling and the reason behind is the production of nitric oxide which has a protective role in the lipid peroxidation caused by freezing and thawing (Srivastava et al., 2000, 2006) of spermatozoa.
In the present study freshly ejaculated Murrah buffalo semen samples of mass motility of +3 or above on a scale of 0–5 were collected and cryopreserved with l-Arginine (1 mM), spermine-NONOate (10 μM) and no additive. Post-thaw sperm motility, viability, membrane integrity and lipid peroxidation were assessed. Cryopreserved samples without any additive showed significantly (P<0.05) lower motility (32.33±1.45%) as compared to the cryopreserved samples treated with l-Arginine (38.33±1.66%) and spermine-NONOate (43.33±1.45%). Sperm viability, membrane integrity and mitochondrial activity were evaluated by eosin-Y staining, hypo osmotic swelling test and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide] assay respectively. Lipid peroxidation was significantly (P<0.05) increased after freeze-thawing in untreated samples and decreased significantly (P<0.05) in spermine-NONOate (3.11±0.08 nmoles/10⁸ cells) and l-Arginine (3.50±0.0⁸ nmoles/10⁸ cells) treated samples. The results therefore, clearly indicate that l-Arginine and spermine-NONOate play important role in protecting the post-thaw sperm motility, viability, membrane integrity and lipid peroxidation status in the cryopreserved Murrah buffalo spermatozoa.

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Subjecting canine semen to the hypo-osmotic test

Abstract

Fertil. Steril.

Exp. Cell. Res.

J. Dairy Sc.

Fertil. Steril.

Fertil. Steril.

Handbook of the laboratory diagnosis and treatment of infertility

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

J. Reprod. Fertil.

The release of glutamic-oxaloacetic transaminase from bovine spermatozoa as a testing method of assessing semen quality and fertility

Biol. Reprod.

Further evaluation of a simple test for determining the integrity of spermatozoa membrane

Clin. Reprod. Fertil.

Analysis of prevasectomy ejaculates by hypoosmotic swelling test

J. Androl.

The conformation of fluid membranes: Monte Carlo stimulations

Science

Hypoosmotic swelling test; results and discussion