Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration, freezing rate and thawing rate on post-thaw semen quality.

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

Risk of Salmonella transmission via cryopreserved semen in turkey flocks.

To investigate the possibility to carry pathogen bacteria in turkey flocks via cryopreserved semen, research was carried out 1) to investigate the microbial contamination of fresh and frozen thawed turkey semen and 2) to evaluate the effect of the freezing-thawing process on the survival of 3 serovars of Salmonella spp. experimentally inoculated in turkey semen. Five pools of semen diluted 4-fold were cooled, added with 8% of dimethylacetamide as a cryoprotectant, and aliquots of 80 muL were directly plunged into liquid nitrogen to form frozen pellets. Mesophilic viable counts, total and fecal coliforms, Enterobacteriaceae, enterococci, Campylobacter spp., and Salmonella spp. were investigated on fresh and thawed samples. Further, 5 pools of diluted semen were each divided into 3 subsamples, inoculated with 7.8 +/- 0.2 log cfu.mL(-1) of Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup, respectively, and cryopreserved before to assess the postthaw viability of Salmonella spp. strains. Fresh semen was highly contaminated by all of the saprophytic bacteria investigated and the cryopreservation process reduced the amount of mesophilic viable count and total coliforms (P < 0.05) and fecal coliforms, Enterobacteriaceae, and enterococci (P < 0.01) by about 1 log cfu.mL(-1). Conversely, neither Campylobacter spp. nor Salmonella spp. were found as endogenous bacteria in semen. In the inoculated semen, both Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup colonies were recovered postthaw, showing a significant reduction of 2.03 +/- 0.28, 3.08 +/- 0.22, and 2.72 +/- 0.23 log cfu.mL(-1), respectively, compared with the fresh semen (P < 0.001). In conclusion, the cryopreservation process allowed us to obtain a low reduction of microbial count both in endogenous saprophytic bacteria and artificially inoculated Salmonella spp. strains; therefore, the possibility of Samonella spp. transmission to flocks through the use of infected cryopreserved semen does exist.

Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

Kallikrein and kininase excretion in football players after psychophysical stress.

In eleven football players and in four football-reserve players urinary kallikrein and kininase activities were determined before and after an official match. The results showed a significant reduction of kallikrein after the match in football players when the football-reserve players were used for comparison (p less than 0.01). Kininase activity appears increased in football players after the match, but not significantly. The Kininase/Kallikrein ratio after the match resulted significantly increased in football players (p less than 0.05) and very significantly compared to the football-reserve players (p less than 0.01).

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.

Isolation and partial characterization of a kallikrein from mouse submaxillary glands.

A simple procedure to obtain relatively large amounts of purified kallikrein from male mouse submaxillary gland is described. Some chemical and biological properties of this kallikrein have been investigated. The enzyme has a m.w. of 32,000 and shows strong BAEE-esterase activity, as well as kininogenase activity. It is partially inhibited by Aprotinin.

Cryopreservation of rabbit semen using non-permeable cryoprotectants: effectiveness of different concentrations of low-density lipoproteins (LDL) from egg yolk versus egg yolk or sucrose.

This study was designed to identify the most effective non-permeable cryoprotectant (CPA) for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. In a second experiment, the performance of the non-permeable CPAs identified as most effective was assessed in vivo by determining reproductive performances. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (Tris-citrate-glucose and 16% dimethylsulfoxide as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1M sucrose, or 15% egg yolk. The semen was loaded in 0.25mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk (P<0.05). Based on the in vitro data, 3 groups of does (n=30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate than LDL and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen.

The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: DIMETHylacetamide versus dimethylsulfoxide.

This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm.

Immunochemical properties of trypsin fragments of human thyroglobulin.

Purified human thyroglobulin (Tg) was enzymatically digested with trypsin. After completion of digestion, the tryptic digest was fractionated by gel filtration on a Biogel A 1.5 m column. Further separation and isolation of the major peak C was carried out on a Sephadex G-75 column. Nine fractions were separated and antigenic properties evaluated by a specific and sensitive radioimmunoassay using a rabbit antiserum to 19S Tg and three different antisera from patients with autoimmune thyroid diseases. The Tg fragments react with both hetero- and auto-antisera. The highest antigenic activity was found on larger fragments, but a fairly good activity was also observed on fragments (C6, C7) with lower molecular weight. Antigenic determinants of Tg differ in individual sera suggesting that different sets of determinants elicit the autoimmune process.

Serial blood T4 and TSH determinations in low birth weight infants. Influence of gestational age, birth weight and neonatal pathology on thyroid function.

Serial blood T4 and TSH determinations were performed on a total number of 352 low birth weight (LBW) infants: 210 healthy newborns and 142 infants affected by respiratory distress syndrome (RDS), sepsis, hyperbilirubinaemia, hypocalcemia, or hypoglycemia. The healthy infants have been divided into three groups in relation to gestational age (less than or equal to 33 wk, 34-36 wk, greater than or equal to 37 wk). Statistical evaluation among groups shows that reduced T4 concentration in LBW infants is strictly related to gestational age, while TSH failed to demonstrate any significant correlation. No significant influence of birth weight on T4 and TSH has been observed. The preterm newborns affected by RDS or sepsis showed a significant reduction of mean T4 concentrations up to 20th day of life when compared with healthy controls similar for gestational age and birth weight. TSH levels are not significantly different. No significant modifications of T4 and TSH have been found related to the other pathological conditions. The significant reduction of T4 and the high incidence of subjects with low T4 values observed in healthy and sick preterm population lend further support to the use of TSH determination for optimum screening of congenital hypothyroidism.

Bovine hemoglobin cross-linked through the beta chains: functional and structural aspects.

2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch. The reaction of bovine Hb with NFPLP leads to a cross-linkage between the beta subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into alpha beta dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross-link of beta chains may be formed between lysine 82 of beta2 and the N terminus of methionine 2 of beta1 or between the two lysine 82 residues of both beta2 chains. Digestion of modified beta globin chains and isolation of the cross-linked peptide have showed that NFPLP cross-links Met-beta2 and Lys-beta82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute.