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.