Incremento de la eficiencia reproductiva de la cría porcina mediante la aplicación de nuevas técnicas avanzadas de análisis de la cromatina espermática y selección por coloide

Resumo

The porcine sector is a crucial part of the Spanish economy. Spain is the European leader in pork production and ranks second worldwide in the export of this meat. For this reason, the pig industry is very competitive in our country, the objective being to produce more and better meat with fewer animals raised, reducing environmental pressure in a market in which costs are increasing due to the implementation of improvements that allow individualized monitoring of animals. Therefore, reproduction techniques such as artificial insemination (AI) are used to reduce the number of animals. Currently, the pig sector faces two significant challenges: the use of antibiotics in insemination doses (due to the bacterial contamination of refrigerated insemination doses, which affects fertility) and the detection of subfertile boars. The uncontrolled use of antibiotics has generated one of the biggest health crises in the world today: antimicrobial resistance. For this reason, a controlled use of antibiotics is requested, only for therapeutic purposes and when strictly necessary. To solve this problem, this Thesis proposes replacing antibiotics in AI doses with a physical method to eliminate bacteria using colloids and centrifugation. Colloids began to be used with two layers of density gradients (DGC), and later, single layer centrifugation (SLC) was used to save time and money. The SLC method has been tested with Porcicoll colloid in swine, where large volumes of ejaculate need to be processed to be useful in the practice. Therefore, it is necessary to evaluate whether a very low colloid density effectively eliminates bacteria when processing a large volume of ejaculate, and its effect on spermatozoa, to establish its practical usefulness to carry out in the swine industry. Therefore, in chapters IV and V, a large volume of sample was processed using the Porcicoll colloid at a low concentration (20 % and 30 %, P20 and P30) to check if it effectively eliminated or reduced bacterial contamination. Sperm recovery, the effect on sperm physiological parameters (chapter IV), and the state of sperm chromatin were evaluated using canonical correlation analysis (CCA), which allowed the multivariate study of the relationships between microbiology and sperm parameters. (chapter V). The results were promising since all bacteria were reduced or eliminated, especially with P30. Compared to the control, P20 and P30 favored maintaining semen quality during storage. Regarding chromatin, an increase in disulfide bridges was observed after storage, improving compaction. The use of CCA proved to be effective in interpreting many variables simultaneously. In a subsequent study (chapter VI), we evaluated the ability of SLC with P20 and P30 to eliminate specific bacteria using a spiking design with relevant bacterial species. The results corroborated the elimination or reduction of the bacterial load after applying SLC, especially with P30, and better sperm quality after storage in the processed samples. The results of this study supported those of chapters IV and V on the applicability of SLC in the pig sector, being an option to process samples of high genetic value. Therefore, replacing antibiotics in insemination doses using the Porcicoll colloid, P30 seems a good option. It has an acceptable sperm recovery and a very efficient reduction of bacteria, which resulted in sperm doses with improved quality. This effect occurred not only due to bacterial removal but also due to a direct impact on the sperm population. The last chapter (VII) delves into the line of study of sperm chromatin in chapters V and VI. The success of AI depends on correct sperm chromatin structure, which is crucial for fertility. For this reason, attempts are being made to apply new methods for detecting alterations in chromatin to detect subfertile boars. The detection of alterations in the chromatin of porcine sperm is especially complex due to its highly compact structure. This Thesis has developed and evaluated techniques to conduct a comprehensive study of chromatin, detecting DNA damage and changes in its structure using methods to assess all variables globally. We used multivariate analysis strategies to evaluate the association between the variables and the effects of various factors. We found that the parameters defining the chromatin damage showed different changes throughout the refrigerated storage of sperm doses. Thus, no correlation was initially observed between reactive oxygen species (ROS) and sperm DNA oxidation, but these variables were correlated after storage. The opposite occurred between the DNA fragmentation and oxidation variables, which showed an initial correlation that disappeared after 11 days. In addition, in this chapter (VII) we performed a principal component analysis (PCA), in which the PC (principal component) that captured most of the variance was fundamentally associated with the disulfide bridge analysis, suggesting that its dynamics may be relevant during refrigerated storage. Furthermore, parameters derived from sperm chromatin structure assay (SCSA) and 8–oxo–7,8-dihydro-2'-deoxyguanosine (8–oxo–dG) may contribute different information about chromatin and perhaps complementary to that provided by disulfide bridges. The combination of the evaluation of SCSA, 8-oxo-dG and disulfide bridges seems to provide a complete picture of the state of the sperm nucleus in boars. Specifically, detecting DNA oxidation with 8-oxo-dG (developed for pigs in this Thesis) and evaluating disulfide bridges could be helpful by detecting variability between males (detection of subfertility) after storage. However, chromomycin A3 (CMA3) was less informative. The results of this Thesis should be tested with fertility studies to obtain robust conclusions about the usefulness of these techniques in improving and predicting the fertility of boars, which would provide helpful and potentially applicable information to the pig sector and would obtain great benefits.