Canine-chilled sperm: study of a semen extender made with low-density lipoproteins from hen egg yolk supplemented with glutamine.

The objective of this study was to evaluate the effects of a combination of 6% low-density lipoproteins (LDL) and 20 mm glutamine in comparison with other extenders used for the refrigeration of canine semen: Tris egg yolk (EY) 20% and 6% LDL. The percentages of mobile spermatozoa after 4 days storage in a domestic refrigerator at +4 °C were 53.1%, 44.2% and 52.2% for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders respectively for 100% of the dogs. After 7 days of storage, these percentages fell to 37.8%, 26.4% and 33.6% in the same extenders for 50% of the dogs. In vitro fertility tests were performed with all of the extenders following the mobility results. These tests were conducted on the day of sampling (D0), and 48 and 96 h after sampling. The results of the hypo-osmotic swelling test were 82.6%, 81.2% and 85.7% on D0, 75.2%, 74.1% and 78.5% on D2, and 70.8%, 71% and 76.1% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. For the FITC/pisum sativum agglutinin (PSA) test, the results were 81.5%, 70.2% and 84.8% on D0, 78.9%, 62.3% and 84.2% on D2, and 72.7%, 59.6% and 73.7% on D4 for the 6% LDL + 20 mm glutamine, 20% EY and 6% LDL extenders, respectively. The acridine orange test was positive; in nearly 100% of cases, none of the spermatozoa had been denatured on D0, D2 and D4. The 6% LDL + 20 mm glutamine and the 6% LDL extenders are capable of preserving spermatozoa that have been stored in a domestic refrigerator at +4°C for at least 4 days. This means that the spermatozoa retain good cytoplasmic membrane integrity, had not capacitated and contained intact DNA in comparison with spermatozoa preserved in the egg yolk extender. The duration of storage is a very important consideration when faced with the problem of sending semen over ever-greater distances.

The advantages of combining low-density lipoproteins with glutamine for cryopreservation of canine semen.

Twenty sperm samples from five dogs were frozen in liquid nitrogen at -196 degrees C in 16 different media, two control media containing 20% egg yolk and 6% low-density lipoproteins (LDL); 10 test media containing 6% LDL (the active cryoprotective ingredient of chicken egg yolk) combined with 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mmol of glutamine respectively at 4%, 5%, 7%, and 8% LDL. Following thawing, sperm mobility was assessed using an image analyser, HAMILTON THORN CERROS 12. The percentage of mobile spermatozoa was 62.05% in the 6% LDL + 20 mmol glutamine medium compared with 48.90% in the egg yolk-based medium (p < 0.05) or 57.55% for the 6% LDL medium (p < 0.05). Furthermore, in most cases, the motility parameters (average path velocity, curvilinear velocity, straight line velocity) in the 6% LDL + 20 mmol glutamine medium, were superior, to a statistically significant extent, to those in the control media. Finally, the 6% LDL + 20 mmol glutamine combination provides spermatozoa with better protection during freezing than egg yolk or the 6% LDL medium alone in terms of acrosome integrity (fluorescein isothiocyanate--Pisum sativum agglutinin test: p < 0.05), the flagellar plasma membrane (hypo-osmotic test: p < 0.05 for 6% LDL), the DNA (acridine orange test; no significant difference) and the integrity of the acrosome (Spermac test: no significant difference).

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen.

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze-thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at -196 degrees C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively. Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p<0.05). In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p<0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test). In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24h; 200x10(6) spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%). In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze-thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone. Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.

Segregation of chromosomes in sperm of a t(X;18)(q11;p11.1) carrier inherited from his mother: case report.

Balanced reciprocal translocations are the most common structural abnormalities; most involve two autosomes while a few involve a gonosome (X or Y chromosome) and an autosome. These rearrangements are usually associated with infertility and/or a higher risk of chromosomal imbalances among offspring. This 26 years old man was first seen because of a 3-year history of primary infertility. He had been found to have a translocation, t(X;18)(q11;p11.1), inherited from his mother when he was 9 years old. Semen analysis showed a very severe oligoasthenoteratozoospermia (OAT). A total of 447 spermatozoa were analysed using three-colour fluorescent in situ hybridization (FISH). The alternate segregation pattern, leading to a normal or balanced chromosomal content, was found in 54.36% of the spermatozoa studied. The frequencies of Adjacent I, Adjacent II, 3:1 segregation and diploidy (or 4:0 segregation) were 8.28, 5.14, 22.37 and 2.01%, respectively. Balanced reciprocal translocations between an autosome and the X chromosome lead to important disruptions in human spermatogenesis. Almost all the males with an X-autosome translocation have azoospermia. The man reported here had very severe OAT and is the first in whom the meiotic segregation pattern was analysed. This case further emphasizes the interest in performing FISH studies in infertile males with a chromosomal translocation to provide them with a personalized imbalance risk.

Value of autoantibodies to beta(2)-glycoprotein 1 in the diagnosis of antiphospholipid syndrome.

OBJECTIVES: To define the specificity and positive predictive value of anti-beta(2)-glycoprotein 1 (anti-beta(2)GP1) antibodies for the diagnosis of antiphospholipid syndrome (APS). METHODS: We determined the presence of anticardiolipin (aCL) antibodies and anti-beta(2)-glycoprotein 1 (anti-beta(2)GP1) immunoglobulin (Ig) G and IgM in 191 consecutive sera from 191 patients and reviewed clinical data separately. aCL IgG and IgM were detected separately using commercial ELISA kits. Anti-beta(2)GP1 antibodies were detected with an in-house ELISA using beta(2)GP1. RESULTS: Seven patients were diagnosed as having APS and 184 as having other diseases. Thirty-six patients were aCL-positive and 12 were anti-beta(2)GP1-positive, seven of these 12 were APS patients. The specificity for anti-beta(2)GP1 in our population was 97%, with a positive predictive value (PPV) of 58%. Among the aCL-positive patients, specificity was 90% and PPV 70-87%. CONCLUSIONS: This study shows that anti-beta(2)GP1 antibodies have a higher specificity and PPV than aCL for APS. The PPV of anti-beta(2)GP1 was greater in aCL-positive than in all patients. We conclude that screening for anti-beta(2)GP1 antibodies in aCL-positive patients increases the specificity and the PPV of aCL testing. In addition, we show that there is no need to screen for anti-beta(2)GP1 antibodies in the absence of aCL antibodies and in the absence of strong clinical suspicion of APS.