A simple method for the quantitative determination of sperm aggregation

Described in this paper are 2 methodological variants for photometric recording of sperm aggregation. Heparin was used to induce aggregation. One of the working principles was related to aggregation-associated alteration of turbidity in an agitated cell suspension, measured by the KZM-1 coagulation-time meter, in analogy to measurement of thrombocyte aggregation. The 2nd variant was based on variation of cloudiness due to sedimentation of aggregates in a non-agitated suspension. Both methods provided equally valid information. They characterised both intensity of aggregation as a combined effect of the number and size of aggregates as well as the rate of aggregation. Photometric recording has proved to be an objectivated method for quantitative assessment of aggregation. Its use is proposed for studies into capacitation or immunological response of spermatozoa.     

Effect of Nitrogen Rate and Stubble Height on Dry Matter Yield,Crude Protein Content and Crude Protein Yield of a Sorghum–Sudangrass Hybrid[Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf.] in the Three‐Cutting System

In this study, the effects of nitrogen (N) rate (60, 120, 180 and 240 kg N ha^?1 applied in three equal dressings at seeding and after the first and second cuttings) and stubble height (7, 14 and 21 cm) on the dry matter (DM) yield, crude protein (CP) content, and CP yield of a sorghum–sudangrass hybrid [Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf., cv. Pioneer 988] in the three‐cut system was investigated. The N rate had no significant effect in the first and third cuttings, but in the second cutting DM yields increased significantly with increase in N rate. The highest yield of 9.1 t ha^?1 was obtained with 80 kg N ha^?1 for the average of 2 years at the second cutting, but no significant difference was found among the 40, 60 and 80 kg N ha^?1 rates. CP content and yield were not significantly affected by N rate at the first and third cuttings, but CP content and yield were significantly affected by application of N at the second cutting. Stubble height had a significant effect on CP content at the third cutting. However, it had no significant effect on CP content at the first and second cuttings. Stubble height had a significant effect on the CP yield at the first cutting, but no significant effect on CP yield at the second and third cuttings.     

Effect of Collection Rhythms and Season on Rabbit Semen Production

The effects of collection regimen and time of year on rabbit semen production were determined in this study. A total of 14 crossbreed Hyla bucks were used in winter and summer. In each season, rabbits were assigned to two groups. In group 1, (n = 7) rabbits were subjected to an extensive collection regimen (two ejaculates per male, once daily/week) and in group 2, (n = 7) a semi‐intensive semen collection regimen was performed (two ejaculates per male, twice weekly). The traits recorded for each sample were libido, volume, pH, motility, sperm concentration, percentage of alive spermatozoa and sperm abnormalities. The results obtained in this study indicate that when increasing collection frequency, the rate of useful collections decreased (from 0.81 ± 0.017 to 0.69 ± 0.016; p < 0.01). The rate of useful collection also decreased in the transition from winter to summer (from 0.79 ± 0.018 to 0.70 ± 0.017; p < 0.01). Among the ejaculate characteristics studied, only volume/ejaculate (from 0.64 ± 0.015 to 0.53 ± 0.017; p < 0.01) and spermatozoa/ml (from 406 ± 15 to 359 ± 13 million; p < 0.01) appeared negatively affected by collection. In winter fewer volume/ejaculates were produced (0.55 ± 0.015 vs 0.60 ± 0.016 ml; p < 0.01) and fewer spermatozoa/ml (360 ± 14 vs 394 ± 16 million; p < 0.01) than in summer. The doses produced per ejaculate decreased as collection frequency increased, but the number of doses produced per week was higher in the semi‐intensive than the extensive rhythm (26.5 ± 2.1 vs 20.9 ± 1.5; p < 0.01). The results suggest that a semi‐intensive rhythm may be viewed favourably.     

Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.