P.M. Zavos

THE HYPOOSMOTIC SWELLING TEST: ITS EMPLOYMENT AS AN ASSAY TO EVALUATE THE FUNCTIONAL INTEGRITY OF THE FROZEN-THAWED BOVINE SPERM MEMBRANE

The objective of this study was to determine the effectiveness of the hypoosmotic swelling (HOS) test together with the supravital test as a means of evaluating the functional integrity of frozen-thawed bovine sperm membrane. A solution consisting of equal parts of fructose and sodium citrate was prepared and the osmolality varied from 50 to 300 mOsm/L. From these various solutions under study, the 100 mOsm/L solution resulted in a maximal number of clearly identifiable swollen spermatozoa. The results from the supravital test indicated that the HOS solution preserved the integrity and prevented excessive lysis of the sperm membrane during the assay. A good correlation was found between the percentage of motile spermatozoa and spermatozoa that reacted to the HOS test (r = 0.73) and between the percentage of sperm with intact membranes and HOS reactive sperm (r = 0.81). Spermatozoa showing swelling of the entire tail region accounted for more than 60% of the total swelling for the HOS solution at 100 mOsm/L. The results obtained in this study indicate that frozen-thawed bovine spermatozoa did react to the HOS test. This technique could prove useful in studies involving the function of the sperm membrane and could possibly predict the sperms ability to fertilize.

Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program

Frozen semen specimens from 22 Holstein bulls representing a wide range of field fertility levels or nonreturn rates (NRR) were used in this study. Semen specimens were thawed at 37 degrees C for a minimum of 30 sec, followed by assessment via a routine semen analysis (RSA) and other sperm functional tests. The RSA was performed by assessing sperm count, motility and morphological characteristics. Other sperm functional tests were performed by assessing the acrosomal membrane integrity, sperm penetration into the cervical mucus and the sperm membrane functional integrity. Following assessment of sperm characteristics, the fertility data of the various bulls were compared to the RSA and the functional tests results. Bulls of high and low fertility were similar in terms of sperm count and progressive motility (P > 0.05). Other characteristics measured by the RSA and functional tests were significantly higher in high fertility bulls (P < 0.05). Correlation coefficients among the various sperm characteristics and fertility of bulls were highly significant (P < 0.01). The highest correlation coefficients between sperm characteristics and fertility were obtained for motility (r = 0.53; P < 0.01), normal morphology (r = 0.59; P < 0.01) and swollen spermatozoa (r = 0.57; P < 0.01). Analysis of specific sperm swelling patterns showed that those patterns considered to reflect maximal sperm swelling were indicative of high fertility.

Preparation and recovery of frozen-thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies

A number of semen manipulative techniques are currently available to remove the undesirable spermatozoa, debris and other factors and to increase sperm quality. The use of motility stimulants such as caffeine or others could optimize the recovery and quality of frozen-thawed spermatozoa processed by a variety of sperm selection techniques. Frozen-thawed specimens from 5 bulls were slowly diluted and washed with Hams F-10 medium containing 3% BSA (w/v) and 0 or 2 mM caffeine. Aliquots containing approximately 50 x 10(6) total sperm cells were used for conventional sperm wash, swim-up, Percoll density gradient centrifugation (80, 70, 55 and 40% Percoll gradients) and Sephadex (SpermPrep I) filtration. Quantitative and qualitative characteristics of selected spermatozoa included: total sperm (x 10(6)), percentage and grade (0 to 4) of motility, percentage of spermatozoa with coiled tails and response to the hypoosmotic swelling (HOS) test (percentage of swollen spermatozoa). When compared to washed specimens, fewer spermatozoa were recovered via the swim-up, Percoll and SpermPrep I filtration methods. Quantitative and qualitative characteristics of these spermatozoa improved further after processing with Hams F-10 containing 2 mM caffeine, followed by selection via the various techniques. Enhancement of sperm motility, in conjunction with the most appropriate sperm selection technique, represents an efficient method for the recovery of spermatozoa with improved qualitative characteristics.

Clinical improvements of specific seminal deficiencies via intercourse with a seminal collection device versus masturbation

In the present study of 50 patients, it was determined that those with seminal deficiencies in masturbated samples showed greater improvement in semen parameters with I-SCD use than the nondeficient groups with which they were compared. The use of I-SCD in oligospermia and OTA Syndrome is therefore indicated, both diagnostically and therapeutically. The subjective rating of sexual stimulation elicited when I-SCD was used was far superior than with masturbation. It is believed the success of I-SCD is due, in part, to this greater degree of sexual stimulation, presumably by added loading of the vas deferens prior to ejaculation.

Effect of feeding tall fescue seed infected by endophytic fungus ( Acremonium coenophialum ) on reproductive performance in male rats

An experiment was conducted to determine the effect of feeding tall fescue seed infected by an endophytic fungus Acremonium coenophialum on the reproductive performance of male rats. Thirty 70-day-old rats were randomly allocated to four treatments: (I) fed 50% rat chow and 50% healthy fescue seed ad libitum (control; N=9; (II) same as I but restricted to the daily feed intake of III (N=7; (III) 50% fungal-infected fescue seed and 50% chow (N=7); and (IV) a mixture of 50% laboratory chow, 25% healthy fescue seed and 25% fungal-infected fescue seed (N=7). The rats were fed these diets for 42 days. During this time, body weights were taken weekly and feed intake was taken daily. At the end of the experiment, the rats were sacrificed and the testes and epididymides were excised and measured. Sperm parameters were assessed (concentration, percentage of motility and progressive motility) at the site of the cauda epididymis; the testes were homogenized and assessed for daily sperm production potential (DSP). Concentrations of spermatozoa (x 10(6)) among the various treatments were 645.6, 486.5, 387.4 and 457.1; motility and progressive motility measurements ranged from 48 to 50% and 2.3 to 2.4 (0-4), respectively. DSP values (per gram) and testicular weight were reduced (P<0.05) in Diet III. The data suggests that 50% fungal-infected fescue seed in the diet of rats does influence the DSP, testicular parechyma and epididymal weight in the rat.

Confocal scanning laser microscopy of morphometric human sperm parameters: correlation with acrosin profiles and fertilizing capacity.

OBJECTIVESnTo develop quantitative criteria for assessing sperm morphology and to determine the correlation between the percentage of morphologically normal spermatozoa and the outcome of the sperm hypo-osmotic swelling test, sperm acrosin profile, and sperm capacity for fertilization.nnnDESIGNnThe maximal length and width of the sperm head, the length of the midpiece and principal piece of the sperm tail, and the ratio of the surface of the acrosomal region to the total surface of the head were determined in specimens obtained from a group of infertile men and a group of fertile men using a confocal scanning laser microscope. Group A consisted of 53 infertile men who were participating in an IVF program, and group B consisted of 98 fertile men. The mean +/- 2 SD of the morphometric parameters in group B was established as representing the lowest and highest normal values in both groups. A normal spermatozoon was defined as one with morphometric parameters within normal levels. The lowest percentage of morphologically normal spermatozoa, hypo-osmotic swelling test result, and acrosin activity in group B were also taken as the lowest normal values in group A.nnnSETTINGnIn vitro fertilization program at the Tottori University School of Medicine, Yonago, Japan.nnnMAIN OUTCOME MEASURESnSperm morphometric parameters, percentage of morphologically normal spermatozoa, hypo-osmotic swelling test, and acrosin activity.nnnRESULTSnThe length of the midpiece, ratio (x 100) of the surface of the acrosomal region to the total surface of the sperm head, percentage of morphologically normal spermatozoa, outcome of hypo-osmotic swelling test, and acrosin activity were significantly higher in group B than in group A. The maximal width of the head was significantly lower in group B than in group A. Strongly positive correlations were observed between percentage of morphologically normal spermatozoa or length of midpiece and the proportion of fertilized oocytes in group A and between ratio (x 100) of the surface of the acrosomal region to the total surface of the head and acrosin activity in groups A and B. Sperm morphology showed high positive and negative predictive values for acrosin activity (normal/abnormal) and fertility potential (present/absent).nnnCONCLUSIONSnUsing quantitative strict criteria, we found that sperm morphology was an important predictor of sperm fertilizing capacity. The confocal scanning laser microscope provided useful information about the sperm cytoskeleton and its importance in fertilization.

Seminal parameters of ejaculates collected from oligospermic and normospermic patients via masturbation and at intercourse with the use of a silastic seminal fluid collection device

Seminal fluid parameters of ejaculates collected via intercourse with the use of a new Silastic seminal fluid collection device (SCD; HDC Corporation, Mountain View, CA) and via masturbation were compared. Thirty couples participated in this study. All males produced two specimens within 8 days with exactly 4 days of sexual abstinence each time. Thirteen patients were classified as oligospermic and the remaining 17 as normospermic on the basis of sperm numbers per milliliter. Each male patient subjectively rated (scale, 0 to 10) his sexual stimulation elicited during production of specimens with the use of the two methods. Specimens were evaluated and compared. Significant increases (P less than 0.05) were observed in all parameters observed within each group of patients when the SCD and masturbation methods were compared. The improvements in sperm parameters because of the method of collection were greater (P less than 0.05) in the oligospermic group than in the normospermic group of patients. The results point out that the SCD, as applied in this study, can assist in the improvement of the collected specimens and produce a specimen that closely resembles the ejaculate obtained during intercourse. In addition, the SCD, as shown in this study, can be of greater assistance to male infertility patients with oligospermia and other spermatogenic dysfunctions.

Effect of feeding fungal endophyte (Acremonium coenophialum )-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding.

This study was designed to assess the effect of endophyte-infected (Acremonium coenophialum ) tall fescue (KY-31) seed (80% infected) on reproductive performance in CD-1 mice by continuous breeding. Twenty-four pairs of 70-d-old CD-1 mice were randomly allocated to four diets: 1) mouse chow ad libitum; 2) 40% infected fescue seed and 60% chow (w/w); 3) reduced intake (100% chow) similar to the intake, adjusted daily, in Diet 2; and 4) 60% infected fescue seed and 40% chow. Males and females were randomly paired (six pairs/treatment) and placed on the above diets. The mice were fed the corresponding diets for 80 d, although the pairs were separated on Day 60 (prior to the birth of the 3rd litter) and the females were monitored for one additional gestation period (20 d). The pregnancy data (litters produced) among the four treatments were 100.0 (18), 77.8 (14), 100.0 (18) and 80.0% (12) respectively. Similarly, the average number of pups born per litter among the four treatments was 11.8, 9.3, 10.1, and 9.8. When the chow treatment (1 and 3) and the fescue treatments (2 and 4) were pooled and compared, the percent pregnancy was 100.0 (n = 36) and 78.8 (n = 26), and the pups born per litter (means +/- SEM) were 11.0 +/- 0.5 and 9.5 +/- 0.6, respectively. Also the intervals between the three litters born during the 60-d cohabitation period were 21.6 +/- 1.1 and 24.5 +/- 0.9 d for the chow and fescue treatments, respectively. The results point out that 40 and 60% infected fescue seed in the diet of mice does influence (P < 0.05) their reproductive capacity as measured by percent pregnancy and litter size.

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.

Human reproductive cloning: the time is near

Reproductive cloning today continues to preoccupy the general public and its critics in a very controversial and often misleading manner. We, in the field of scientific and reproductive medicine, realize that our responsibilities are quite numerous and extremely delicate. It was not too long ago that we witnessed the atmosphere at the National Academy of Sciences hearing in Washington, DC (August 2001) on the topic of human reproductive cloning, although not entirely militated against by its concomitant scholarly document (National Academy of Sciences, 2002; Simpson and Edwards, 2003). As one of the invited participants, it was evident from the behaviour of the NAS members and their invited guests that this hearing was scheduled not to discuss the topic of human reproductive cloning, but rather to condemn it. From the beginning of our efforts, we have never stated that we intended to create the first cloned embryo and the first human being for reproductive purposes by ignoring the public’s concerns and the scientific critics. We also never intended to ignore the contradictory results that scientists in the field of animal cloning have obtained during the past years. We merely wanted to learn from all the difficulties that the animal cloning experts encountered, in order to take the criticisms and the public’s concerns as seriously as possible and turn them into positive developments. It was quite evident to us from the beginning of this debate that with further elucidation of the molecular mechanisms involved during the processes of embryogenesis, careful tailoring of subsequently