Sperm Survival Rate Research Articles

Laminaria japonica polysaccharides on the recovery of rats spermatogenic function of testis damaged by chronic local ionizing radiation

To investigate the degree of recovery by Laminaria japonica polysaccharides (LJP) on the damage to the rat spermatogenic function caused by chronic local ionizing radiation, 56 wistar rats were randomly divided into seven groups: Control, three model and three LJP groups. Results showed that although indicators in LJP and model groups were found different from the control group (P 0.05). In the other 7 and 14d LJP group, the SOD and GSH-PX activities increased while MDA content decreased. In addition, mitochondrion membrane potential enhanced in testis cells while DNA injury relieved; furthermore the sperm count and survival rate increased, the difference was significantly (P < 0.05) and the testicular tissue damage reduced and mitochondrion were relieved, which were more similar to those of the control group. Our results suggested that lavaged by LJP100 mg·kg-1·d-1 at the duration from 7 days before radiation till the end of radiation, LJP has promoting effects on the recovery of the damages to spermatogenic function induced by chronic local ionizing radiation. Key words: Laminaria japonica polysaccharides, ionizing radiation; spermatogenic function, damage recovery.

Effects of activating mediums with different salinity, potassium ion and conservation time on sperm motility of Anguilla japonica

Sperm motility of Anguilla japonica treated with six activation mediums with different salinities(34,32,30,28,26 and 22),three extender solution with different potassium ion concentration(25 mmol/L,30 mmol/L,35 mmol/L) and different conservation time(0 h,24 h,48 h,72 h and 96 h) was observed and measured.The results showed that: when activation medium salinity was 30,both sperm motility and percentage of motile sperms were the highest,meanwhile the curvilinear velocity(VCL),the straight line velocity(VSL) and the angular path veloc-ity(VAP) were the highest.It could be concluded that the spawning salinity of Anguilla japonica may be 30 in natural environment.When potassium ion concentration in extender solution was 30 mmol/L,both sperm motility and percentage of motile sperms were the highest,meanwhile the curvilinear velocity(VCL),the straight line ve-locity(VSL) and the angular path velocity(VAP) were markedly higher than those under other concentration lev-els(P0.05).So the best osmotic pressure of extender solution may be 796.68 kPa.As the conservation time ex-tended,sperm motility reduced gradually.When potassium ion concentration in extender solution was 30 mmol/L,conservation time of sperm could reach 96 h and sperm survival rate was higher than in 25 mmol/L and 35 mmol/L potassium ion.

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 µl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.

Regulation of fertilization in male rats by CatSper2 knockdown

Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/or openers of these ion channels could interfere with sperm function. In this study, in vivo electroporation (EP) and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower (P<0.05) hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca(2+) peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hyperactivation.

Effect of Uremia on Semen Quality and Reproductive Function in Humans

In this study, we sought to evaluate the effect of uremia on semen quality and reproductive function in humans. For this purpose, 53 end-stage uremic patients were randomly selected. The semen samples were produced by masturbation. Fertility index (FI) was calculated according to the following formula: sperm density (×10(6)/ml) × sperm motility (%) × normal sperm morphology rate (% per 10,000). The semen samples of uremic patients were compared with those of fertile and infertile males. The results show that three patients failed to produce semen. There were no sperm found in four semen samples. The sperm motility, survival rate, sperm density, and normal sperm morphology rate of the remaining 46 patients were found to be significantly lower than those of controls. The uremic patients had the FI of 0.68(2.08) which was obviously lower than that of fertile 7.7(13.51) and infertile 4.13(5.77) males. It was, therefore, concluded that uremia caused a significant decline in sperm quality and reproductive function which resulted in consequential infertility in humans.

Effect of ejaculation frequency on spermatozoa survival in diluted semen from Pleven Blackhead rams

The effect of ejaculation frequency on spermatozoa survival in diluted Pleven Blackhead ram semen was investigated. Thirty-two ejaculates were examined. From each ram 4 ejaculates were collected at 10-min intervals in the morning and in the afternoon. Semen parameters, namely, color, density, transparency, volume, concentration, motility, and abnormal sperm, for each ejaculate were determined. The sperm motility was assessed from the moment of dilution up to 60 min at 10-min intervals, then again at 90 min and 120 min. The semen characteristics were determined by means of "Motic Image Plus" equipment and the data were processed statistically. Sperm volume and concentration in semen samples decreased gradually with increase in ejaculation frequency. The values were significantly lower for the 4th ejaculate compared to preceding ones (P < 0.05). The highest survival rate (P < 0.05) of sperm was found in the 2nd ejaculate, and this trend was preserved up to the second hour. Up to 50 min after dilution, the sperm motility did not change considerably, after which it was found to be reduced (P < 0.05). The frequency of ejaculation and the period of semen collection had an impact on sperm motility in extended and short-time stored semen from Pleven Blackhead rams. The spermatozoa in a 2nd ejaculate showed higher survival rates and could be recommended for additional processing.

Assessment of Motility and Survival Rate of Asthenospermic Men's Sperm Cultured in Media Plus Leukemia Inhibitor Factor

Assessment of Motility and Survival Rate of Asthenospermic Men's Sperm Cultured in Media Plus Leukemia Inhibitor Factor

Semen characteristics of boars receiving control diet and control diet supplemented with L-carnitine

The objective of this experiment was to test a hypothesis that L-carnitine supplemented diet would improve semen characteristics in 6 adult crossbred AI boars (Hampshire × Pietrain). The control and the tested diet were identical except the tested period (8 weeks) when the diet was supplemented with 2 g of L-carnitine per boar per day. The semen was collected regularly weekly by a gloved-hand technique. Semen volume, sperm motility and concentration, proportion of alive sperm cells and aspartate aminotransferase (AspAT) activity of semen were determined immediately after the semen collection once every two weeks. Sperm survival rate, morphologically abnormal spermatozoa, seminal plasma mineral components and free amino acid concentration, L-carnitine concentration in semen plasma and in sperm cells were determined after the sample storage (–20°C) at a later time. The differences ascertained between the average values of semen characteristics in the control vs. tested period did not prove a true and unambiguous positive effect on boar semen parameters by dietary supplementation of L-carnitine as our data show in our study: volume (239.11 vs. 250.50 ml; &lt;I&gt;P&lt;/I&gt; 0.518), sperm concentration (301.67 vs. 350.83 × 10&lt;sup&gt;3&lt;/sup&gt;/mm&lt;sup&gt;3&lt;/sup&gt;; &lt;I&gt;P&lt;/I&gt; 0.309), sperm progressive motility (66.94 vs. 70.00%; &lt;I&gt;P&lt;/I&gt; 0.409), morphologically abnormal spermatozoa (29.00 vs. 27.46%; &lt;I&gt;P&lt;/I&gt; 0.802), daily sperm cells output (9.86 vs. 11.71 × 10&lt;sup&gt;9&lt;/sup&gt;; &lt;I&gt;P&lt;/I&gt; 0.206), proportion of alive sperm cells (72.56 vs. 74.13%; &lt;I&gt;P&lt;/I&gt; 0.484), survival spermatozoa ability maintenance (43.29 vs. 38.68%; &lt;I&gt;P&lt;/I&gt; &lt; 0.01), mineral components in the seminal plasma (Na-, K-, Ca-, Mg-, Zn-; P from 0.138 to 0.968), AspAT activity (in semen plasma – 132.50 vs. 128.31 mU/10&lt;sup&gt;9&lt;/sup&gt; spermatozoa; &lt;I&gt;P&lt;/I&gt; 0.830, in sperm cells – 147.37 vs. 119.01 mU/10&lt;sup&gt;9&lt;/sup&gt; spermatozoa; &lt;I&gt;P&lt;/I&gt; 0.146), semen plasma amino acid concentration – a significant positive effect of L-carnitine in lysine only (0.79 vs.1.17 &amp;mu;mol/100 ml; &lt;I&gt;P&lt;/I&gt; &lt; 0.01), L-carnitine concentration (in semen plasma 255.40 vs. 259.97 mg/l; &lt;I&gt;P&lt;/I&gt; 0.884, in sperm cells – 1 110.68 vs. 883.58 mg/l; &lt;I&gt;P&lt;/I&gt; &lt; 0.01). In conclusion, the studied indicators of semen quality were not significantly enhanced by dietary supplementation of L-carnitine in adult AI boars.

Mating duration and egg production of the predaceous mite Neoseiulus californicus (Acari: Phytoseiidae) vary with temperature

Effects of constant temperature on mating duration and total fecundity of Neoseiulus californicus females mated once were investigated at 18 °C, 25 °C, 30 °C, and 35 °C with a photoperiod of 16L:8D. Adult mites grown and maintained at 25 °C mated for 315.3 min on average and produced 46.1 eggs per female. These values varied significantly by temperature: 553.6 and 13.9 (18 °C), 261.2 and 26.6 (30 °C), and 253.6 and 23.9 (35 °C), respectively. Duration of copulation was negatively correlated with temperature. However, total egg production peaked at 25 °C and decreased at lower and higher temperatures. Reduced sperm transfer and/or survival rate of sperm in the female body may account for decreased egg production when temperatures are not optimal.

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield, motility and survival

A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4 degrees C overnight, were also available for testing. For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18-20% when cold-stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.

Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post-thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing-resistant (or intact) and eliminating non-viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo-methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen-thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post-thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo-injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.

Effect of Ultraviolet Irradiation on Sperm of the Left‐eyed Flounder, Paralichthys olivaceus

Abstract In this study, at proper dosage of ultraviolet (UV) irradiation (180 sec: 36,000 erg/mm2), sperm chromosomes of left‐eyed flounder, Paralichthys olivaceus, were inactivated, while spermatozoa maintained ability to move and inseminate eggs. Gynogenetic haploids were detected by morphological observation, chromosome counting, and flow cytometer analysis. The ultrastructure of treated sperm was observed under scanning electronic microscope (SEM) and transmission electronic microscope (TEM). The results showed that after being irradiated at lower dosage of irradiation (0–180 sec: 0–36,000 erg/mm2), the surface structure of spermatozoa was not affected by UV irradiation, while the inner structures including membrane system and karyoplasm denseness of treated spermatozoa were little changed. However, obvious changes were observed in their membrane system, mitochondria, and nucleus if the dosage of irradiation increased to 240 sec: 48,000 erg/mm2 or 300 sec: 60,000 erg/mm2. The sperm survival rates did not change at the lower dosages of the UV irradiation (0–180 sec: 0–36,000 erg/mm2) but decreased as the irradiation dosage increased. The motility of treated sperm was lower than that of control group in general but did not change with UV irradiation dosage increasing at the certain range of 0–300 sec: 0–60,000 erg/mm2.

Targeted disruption ofSlc2a8(GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8+/+, Slc2a8+/−, and Slc2a8−/− mice, respectively) resulting in a deviation (p = 0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.

224 A COMPARATIVE STUDY BETWEEN WOOD AND PLAINS BISON

In Canada, brucellosis and tuberculosis threaten an estimated 4500 wood bison (Bison bison athabascae), a species considered at risk by the Committee on the Status of Endangered Wildlife In Canada (COSEWIC). To help rescue this species, our Wood Bison Reproductive Research group proposes to employ advanced reproductive technologies. Unfortunately, little is known about the reproductive physiology of the wood bison, which hinders the application of these reproductive technologies. In order to modify advanced reproductive techniques developed in cattle for use in wood bison, the large amounts of semen, embryos, and oocytes from wood bison required are not available. The purpose of this study was to compare semen collected from the more abundant and closely related plains bison (Bison bison bison) with that of wood bison. Semen from 3 wood and 4 plains bison were collected by electro-ejaculation during the summer of 2007. Andrological parameters of morphology and motility were recorded on fresh semen, extended semen, and post-thawed semen samples. A Student's t-test was used to compare the results of these two groups. Semen was cryopreserved using two commercially available cryopreservation media (Andromed and Triladyl, Minitube Canada, Ingersoll, Ontario, Canada). Sperm morphology and motility were not different between electro-ejaculated samples from plains and wood bison (P &gt; 0.05). Also, no difference was found in the survival rate of sperm from the electro-ejaculated samples between plains and wood bison after freezing and then thawing using an egg-yolk based extender (Triladyl) or an extender containing no products of animal origin (Andromed). A difference between cryopreservation media was found; post-thaw motility of Triladyl-treated sperm was higher (29%) than that of the Andromed-treated sperm (12%). Due to lack of previous success with preserving electro-ejaculated semen in media free of animal-origin products, motility assays were performed to evaluate if spermatozoa retrieved from epididymides of plains bison can be cryopreserved in Andromed. Interestingly, cyropreserved epididymal spermatozoa had a higher motility than cryopreserved electro-ejaculated sperm after freezing-thawing procedures using a medium containing no products of source animal (respectively, 30% v. 7%). This result suggests that there may be a factor secreted by the reproductive accessory glands that interferes with the post-thaw survivability of bison sperm. In conclusion, this study supports the hypothesis that semen from plains bison behaves similarly to that of wood bison semen during cryopreservation and therefore could be used to establish protocols for advanced reproductive technologies in wood bison. This project was supported by Canadian Adaptation and Rural Development in Saskatchewan.

Seminal fluid signaling in the female reproductive tract: Lessons from rodents and pigs1

Seminal fluid contains potent signaling agents that influence female reproductive physiology to improve the chances of conception and pregnancy success. Cytokines and prostaglandins synthesized in the male accessory glands are transferred to the female at insemination, where they bind to receptors on target cells in the cervix and uterus, activating changes in gene expression that lead to modifications in structure and function of the female tissues. The consequences are increased sperm survival and fertilization rates, conditioning of the female immune response to tolerate semen and the conceptus, and molecular and cellular changes in the endometrium that facilitate embryo development and implantation. Male-female tract signaling occurs in rodents, livestock animals, and all other mammals examined thus far, including humans. In mice, the key signaling moieties in seminal plasma are identified as members of the transforming growth factor-beta family. Recent studies indicate a similar signaling function for boar factors in the pig, whereby the sperm and plasma fractions of seminal fluid appear to synergize in activating an inflammatory response and downstream changes in the female tract after insemination. Seminal plasma elicits endometrial changes, with induction of proinflammatory cytokines and cyclooxygenase-2, causing recruitment of macrophages and dendritic cells. Sperm contribute by interacting with seminal plasma factors to modulate neutrophil influx into the luminal cavity. The cascade of changes in local leukocyte populations and cytokine synthesis persists throughout the preimplantation period. Exposure to seminal fluid alters the dynamics of preimplantation embryo development, with an increase in the number of fertilized oocytes attaining the viable blastocyst stage. There is also evidence that seminal factors influence the timing of ovulation, corpus luteum development, and progesterone synthesis. Insight into the molecular basis of seminal fluid signaling in the female reproductive tract may inform new interventions and management practices to ensure maximal fertility and reduce embryo mortality in pigs and, potentially, other livestock species.

Effects of Taurine on Sperm Characteristics during In vitro Storage of Boar Semen

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations oftaurine (25-100 mM) with or without 250 μM H 2 O 2 . The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group (85.1±0.5 and 72.4±0.3, p<0.05). The sperm motility in taurine with H 2 O 2 after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with H 2 O 2 (75.3±0.3 and 69.6±2.9, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM (76.0±0.6) and 50 mM taurine groups (78.0±0.7), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (≥48% in 9 h and ≥42% in 12 h) compared to controls (43.0±2.1 and 31.0±0.6, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics (motility, viability, survival and membrane integrity).

Gene Transfer to Mouse Embryos by Sperm Mediated Gene Transfer Method

Furuta, H., Ichikawa, E., Sugimura, S., Kikuchi, S., Yoshida, T., Mukouyama, H. and Tomogane, H. 2006. Gene transfer to mouse embryos by sperm mediated gene transfer method. J. Appl. Anim. Res., 29: 113–116. To obtain transgenic mouse embryos by in vitro fertilization transgenic mouse sperm were produced by electroporation using green fluorescent protein (GFP)gene as a marker. The sperm was cultured in medium GFP gene. Electoroporation was carried out under 200 V-1μF, 200V-25μF and 200V-50μF. The survival rate of sperm decreased by each treatment. Although the DNA transfer into sperm mediated by electroporation was very easy, efficiency of introduction into embryos was very low. Sperm mediated gene transfer may be inferior to microinjection.

Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm

The response of sperm to cryopreservation and the fertility of frozen-thawed semen varies between species. Besides species differences in sperm physiology, structure and biochemistry, factors such as sperm transport and female reproductive tract anatomy will affect fertility of frozen-thawed semen. Therefore, studying differences in sperm cryotolerance between breeds and individuals instead of between species may reveal sources of variability in sperm cryotolerance. In the present study, the effect of cooling, re-warming and freezing and thawing on plasma membrane and acrosome integrity of sperm within and between Norwegian Landrace and Duroc breeds was studied. Furthermore, the relation between post-thaw survival rate and fatty acid composition of the sperm plasma membranes was investigated. Flow cytometry assessments of plasma membrane and acrosome integrity revealed no significant differences between breeds; however there were significant male-to-male variations within breeds in post-thaw percentages of live sperm (plasma membrane intact). The most abundant fatty acids in the plasma membranes from both breeds were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5, n-6) and docosahexaenoic acid (22:6, n-3). The ratio of sigma operator 22:5, n-6 and 22:6, n-3/ sigma operator all other membrane fatty acids was significantly related to survival rate (plasma membrane integrity) of sperm for both Norwegian Landrace (correlation coefficient (r(s)) = 0.64, P < 0.05) and Duroc (r(s) = 0.67, P < 0.05) boars. In conclusion, male-to-male differences in sperm survival rate after freezing and thawing may be partly related to the amount of long-chain polyunsaturated fatty acids in the sperm plasma membranes.

105 ALTERING SEMEN EXTENDER AND GLYCEROL CONCENTRATION TO OPTIMIZE RESULTS AFTER CRYOPRESERVATION OF EQUINE SPERMATOZOA

After cryopreservation, spermatozoa from many stallions may have a lower capacity to fertilize an oocyte than fresh or cooled semen. The aim of this study was to evaluate a standard panel of semen extenders and varied concentrations of the cryoprotective agent (glycerol) to optimize sperm survival rates after cryopreservation. Semen was collected from Quarter Horse stallions (n = 3) from March to May 2006 (6 collections per stallion). Semen was filtered immediately after collection, and sample volume and sperm concentration were measured. A drop of raw semen was placed on two prewarmed slides to estimate the percentage of progressively motile sperm. The semen sample was then diluted to 100 � 106 spermatozoa/mL with a dried skim milk glucose extender (EZ Mixin Original Formula; ARS, Chino, CA, USA) or a chemically defined, milk-free diluent (INRA 96; IMV, Maple Grove, MN, USA). After 1 h of slow cooling and equilibration to 4�C, semen samples were centrifuged for 10 min at 400g. A defined volume of supernatant was removed, so that a concentration of 1000 � 106 spermatozoa/mL was obtained after resuspension of the sperm pellet. A 150-�L aliquot of semen was then added to specified quantities of the same semen extender used after semen collection and cryopreservation medium (Cryoguard�; Minitube, Verona, WI, USA) to obtain final glycerol concentrations of 2, 3, and 4%. This also gave a concentration of 100 � 106 spermatozoa/mL. After equilibration for 1 h at 4�C, spermatozoa were loaded into 0.5-mL straws and frozen in liquid nitrogen vapor. After 10 min, straws were plunged into liquid nitrogen. Semen was thawed at 37�C for 30 s and evaluated as prior to cryopreservation. Mean total semen volumes were 56, 11, and 60 mL in the 3 stallions. Their respective mean sperm concentrations were 124 � 106, 505 � 106, and 161 � 106 sperm/mL, respectively. Mean percentages of progressively motile sperm prior to cryopreservation were 64, 89, and 72%, respectively. With the paired Student&amp;apos;s t-test, percentages of progressively motile sperm after cryopreservation were evaluated with respect to semen extender and concentration of glycerol used. Mean overall progressive motility of spermatozoa after cryopreservation differed significantly between the two extenders and was 46% for INRA 96 and 35% for EZ Mixin OF (P &amp;lt; 0.001). Using EZ Mixin OF as semen extender, the best mean post-thaw progressive motility was achieved with 4% glycerol (39%) and differed significantly from that with 2% glycerol (32%; P &amp;lt; 0.01). When INRA 96 was used (49% for 4%, and 42% for 2% glycerol), there was no difference. This results provide evidence that, during freezing of equine spermatozoa, there is a significant effect of the semen extender and the concentration of the cryoprotectant on post-thaw sperm motility. We therefore suggest mini-freezing trials prior to freezing large numbers of sperm to find the semen extender and glycerol concentration that provides optimal survival rates.

Sperm DNA Damage as Measured by the Sperm Chromatin Structure Assay (SCSA) Does Not Predict Sperm Survival Rate

The percentage of sperm with high levels of DNA damage, the DNA fragmentation index (% DFI), has been shown to correlate with abnormal semen parameters and reduced pregnancy outcome in IVF. Sperm survival rates, the percentage of sperm that maintain their motility after sperm processing and 24h incubation, have also been shown to be predictive of IVF outcome. Thus, we designed a study to evaluate the relationship between sperm survival test results and sperm DNA damage anticipating that they would be highly correlated.