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Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10-24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17-22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.

Simple Summary: It remains unclear whether the prolonged preservation of cells, such as sperm, in liquid nitrogen (LN2) leads to their deterioration over time. In this study, we investigated whether the conservation of Iberian pig semen for ten years results in a decline in quality and whether the evaporation of LN2 from the tanks between refills could be a contributing factor. Furthermore, the only way to improve already-frozen samples is to implement specific thawing protocols. Thus, our findings suggest that only samples consistently submerged in LN2 benefit the most from the application of thawing protocols, such as rapid thawing at 70 °C for 8 s. While partial conservation in LN2 and LN2 vapors does not result in a loss of quality over time, it does cause damage that thawing protocols cannot mitigate. This finding suggests that, independently of the lack of the effect of the level of storage in the tank, careful handling and optimization of thawing protocols are essential for the quality preservation of the cryopreserved sperm samples., The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN2) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN2 levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN2 levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN2, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN2. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN2.

Thawing protocols have been barely studied, and their modifications may lead to a substantial improvement in post-thawing sperm quality, which could be of great relevance to existing sperm banks, such as those for Iberian pig breeds with varieties in danger of extinction. For that, the study aimed to evaluate different thawing rates and to evaluate modifications in the composition of the thawing extender (basic pH to 8-8.2, incorporation of cyclodextrins loaded with cholesterol [CLC] and the incorporation of ion chelators [EDTA and EGTA]). After thawing, overall sperm motility and kinematic parameters, acrosome status and sperm membrane integrity were evaluated. The most optimal results were obtained with the thawing rate reaching 70 °C for 8 s with the inclusion of 12.5 mg of CLC/500 × 106 spermatozoa in the thawing extender, which showed an improvement compared to the control at 70 °C. In conclusion, to adapt the thawing conditions may be relevant, especially for endangered species or breeds such as some varieties of Iberian pig, since this process could also be used in samples cryopreserved in gene banks.

With the purpose of improving aquaculture sustainability, the search for protein alternatives to fishmeal makes it necessary to test different variables and the possible repercussions of new ingredients. The use of insect meal as a protein source for aquaculture is well described, but the complex composition of insect meals (fat and other components) can affect the physiology of fish. For this reason, as a part of a bigger study, the aim of the current manuscript was to test diets based on three different presentations of insect meal coming from yellow mealworm (Tenebrio molitor): full fat, partially defatted, and supplemented with a long chain omega–3-enriched oil, and to evaluate their effects on protein digestibility, biometric indices, immunological system and gut health (intestinal histomorphology and microbiota) of rainbow trout (Oncorhynchus mykiss). Digestibility of the protein and body indices showed a minor but consistent trend. The non-specific immunological system did not show changes, but the histology of the intestine showed signs that insect meals could be softening a mild inflammatory response. The gut microbiota suffered several changes, which could be associated with the different amino acid and fatty acid compositions of the diets.

Aquaculture struggles with sustainability due to the use of fishmeal, and insects are one potential alternative. Insects have low long-chain omega−3 polyunsaturated fatty acid content, and insect-fed fish reflect this in their composition. In total, 500 rainbow trout (Oncorhynchus mykiss, ~46 g) were fed until the fish reached a commercial size (~415 g). Five diets were used: one control based on fishmeal as main source of protein; two with a 50% replacement of fishmeal with yellow mealworm meal (Tenebrio molitor), one with full-fat insect, and another partially defatted; two other diets similar to the one with the full-fat insect, but with the addition of an experimental algal oil rich in omega−3 in two different concentrations (one equivalent to control, the other one to the defatted insect diet). Growth was unaffected, as well as texture and organoleptic profile of the fillets. Lightness, brightness and colour of the fillets were slightly modified by the experimental diets. An increase in omega−3 levels over those of the full-fat insect diet is described. An omega−3 sparing effect was highlighted, causing lipid accumulation in fillets and liver, and a mild increase in oxidative damage. More research is encouraged to evaluate the fatty acid profile of the liver.

[EN] The objective of the study was to evaluate the impact of different concentrations of dietary red beet and betaine on the growth performance and fish flesh quality of rainbow trout. Therefore, a control diet was compared with four diets in which two levels of red beet (14% and 28%) and betaine (0.9% and 1.63%) were incorporated in combination. The study was set up with an average body weight of 69 ± 2.2 g and finished when fish reached commercial weight (175¿250 g) after 105 d. The impact of the diets was studied based on the growth performance, biometric indexes, proximal composition, protein and fat retention efficiencies and apparent nutrient digestibility by fish reared on a recirculation system. Further estimates were the effect of red beet and betaine on the flesh proximate composition and quality of the final product (water activity, colour, texture, thiobarbituric acid reactive substances and sensory characteristics). Results showed that inclusion of 14% red beet and 0.9% betaine did not affect growth, nutritive or biometric parameters and nutrient retention when compared with the control diet. However, higher levels of red beet and betaine had negative effects on growth and nutritive parameters. The tested ingredients enhanced quality parameters regardless of the concentration used. After feeding the red beet and betaine, fish flesh showed lower water activity and better textural and colour properties than the control and also a dose-dependent effect on lipid oxidation was observed.

An experiment was conducted to evaluate the replacement of soybean for winter peas with different levels of protease inhibitors, both trypsin and chymotrypsin, on the performance of fatty pigs. Were used a total of 192 Duroc hybrid barrows, with 61 days-old (22.1 kg), which were slaughtered with 167 days of age (121.0 kg). There were three varieties of peas in diets Cartouche (CAR), Iceberg (ICE) and Luna (LUN), with 9.87, 5.75 and 12.55 trypsin-inhibited units (UTI/mg) and 10.16, 8.62 and 15.75 chymotrypsininhibited units (CIU/mg), respectively. The design, in completely randomized blocks, had four treatments according the pea included in the diet control (with soybean), CAR, ICE and LUN diets, with 12 replicates of four pigs per treatment. At the end of the experimental period, pigs fed the control diet were heavier (P < 0.01) and showed higher daily weight gains (P < 0.01) and daily feed intake (P < 0.05) than those fed CAR and LUN diets, with those that received the ICE diet in an intermediate position. These results are mainly due to the first periods (from 61 to 83 days of age), because growth performances were scarcely affected later. No influence of the diet was observed on carcass and meat quality (P > 0.10). It concludes that winter peas with 5.75 TIU/mg and 8.62 CIU/mg, can replace soybean from 84 to 167 days of age, without affecting yield, carcass quality, main lean cuts and intramuscular fat loin content and major fatty acids proportion (C160, C180, C181n-9) of subcutaneous fat. © 2017, Asociacion Interprofesional para el Desarrollo Agrario. All Rights Reserved.

BACKGROUND During freezing the selective precipitation of substances in the medium may provoke a pH shift and lead to sperm damage. OBJECTIVE To study the effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality. METHODS A total of 15 ejaculates from different boars were obtained and divided into six aliquots prior to a standard straw cryopreservation in freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) with different pH. After thawing, sperm quality (plasma membrane integrity, motility and acrosome status) were assessed at 30 and 90 minutes of post-thaw incubation at 37 degree C. RESULTS When the boar sperm were frozen in a freezing media with pH basic, and particularly at pH 8, it had higher post-thaw sperm quality. CONCLUSION The pre-adjustment at pH 8 of the freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) is able to improve the post-thaw boar sperm quality.

A total of 192 Iberian females (Duroc*Iberian), with 110 days of age and 45 ± 3.41 kg BW, were used to study the effect of immunocastration in yield of carcass and prime lean cuts. There were three treatments: surgically castrated females (CC), entire females (CE), and inmunocastrated females (CI) the experimental design was a com- plete randomized blocks with 48 replicates total (4 pigs in each), 12 replicates per block and 16 per treatment. The feed, the same for all animals, was suministered ad libitum. After sacrifice, 31 pigs were selected for each treatment to assess the performance of carcass and main lean cuts. Treatment CI tended to improve slaughter and carcass weights (p<0.10), with the same weight of hams and forelegs that CE (p>0.05) and significantly less in the CC (p<0.05). By contrast, the loins were heavier in CE than in CI and CC (3.05 vs. 2.82, 2.71 kg, respectively; p<0.05). Regression equations were established relating the carcass weight with hams, forelegs and loins weights. The best relationships between variables were obtained with linear functions in the case of ham and foreleg (R 2 >55 %; p<0.05). For loin, linear and non-significantly in CC (p>0.05) and quadratic for CE and CI (p<0.05). The immunocastration favors the growth of Iberian female; the surgical castration is the least interesting option., Se utilizaron 192 hembras Ibéricas (cruzadas, Duroc*Ibérica), con 110 días de edad y 45 ± 3,41 kg de peso vivo para estudiar el efecto de la inmunocastración en el rendimiento de la canal y de las piezas nobles. Los animales experimenta- les se distribuyeron en 3 tratamientos: hembras castradas quirúrgicamente (CC), hembras ente- ras (CE) y hembras inmunocastradas (CI), siendo el diseño experimental en bloques completos al azar, con 48 réplicas totales de 4 cerdas cada una, 12 réplicas por bloque y 16 por tratamiento. El pienso fue el mismo para todos los animales, suministrándose ad libitum. Una vez realizado el sacrificio de los animales, se seleccionaron 31 hembras por cada tratamiento para evaluar la canal y el rendimiento de las piezas nobles. Las cerdas CI tendieron a presentar un mayor peso de sacrificio y peso de canal (p<0,10), siendo los pesos de jamones y paletas similares a las hem- bras CE (p>0,05), y significativamente menos pesados en el grupo de las hembras CC (p<0,05). Por el contrario, el peso de los lomos resultó significativamente mayor en las hembras CE que en las CI y CC (3,05 vs. 2,82; 2,71 kg, respectiva- mente; p<0,05). Se calcularon las ecuaciones de regresión relacionando el peso de la canal con el peso del jamón, la paleta y el lomo para cada tratamiento. Las mejores relaciones entre las va- riables se obtuvieron con funciones lineales en el caso del jamón y la paleta (R 2 >55 %; p<0,05) y, en el caso del lomo, lineal y no significativa en el grupo CC (p>0,05) y cuadráticas para los grupos CE y CI (p<0,05). La inmunocastración favorece el cre- cimiento de las cerdas y que la castración quirúr- gica no presenta ninguna ventaja productiva.

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10 °C and 5 °C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P< 0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P> 0.05), but treatment with 25% did not show significant differences between the time of incubation at 37 °C after thawing (P> 0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480. min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS progressive motility spermatozoa. P< 0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process. © 2012 Elsevier B.V.

En ganado porcino, los resultados de fertilidad (fertilidad y tamaño de camada) obtenidos con semen congelado no son equiparables a los obtenidos con semen refrigerado (a 15-17ºC). Durante los procesos de congelación y de descongelación, los espermatozoides sufren modificaciones que en último término van a provocar una alteración de su funcionalidad. El proceso de crioconservación espermática produce una desestabilización de la membrana en la que se ha observado una pérdida de colesterol. En especies sensibles al choque térmico (incluida la especie porcina), se ha observado una mejora en la calidad del semen post-descongelación, cuando los espermatozoides son incubados con ciclodextrinas saturadas de colesterol (CLC) antes del proceso de crioconservación. El objetivo de esta tesis ha sido determinar cómo afecta este tratamiento pre-congelación con CLC a la funcionalidad in vitro y fertilidad in vivo e in vitro de los espermatozoides congelados-descongelados de la especie porcina. La funcionalidad espermática in vitro se evaluó mediante los siguientes parámetros: resistencia osmótica, movilidad e integridad de la membrana plasmática, estado y dinámica de capacitación, capacidad de adherencia a células epiteliales del oviducto y dinámica de la integridad de la cromatina. Para ello se utilizó un sistema computerizado de movilidad, y se pusieron a punto diferentes técnicas de citometría de flujo y manejo in vitro de cultivos celulares. La fertilidad in vitro se evaluó mediante un test de penetración de ovocitos inmaduros (recuperados de ovarios de cerdas en matadero) y la fertilidad in vivo se evaluó en cerdas que recibieron un tratamiento de sincronización de celo (con PMSG) e inducción de la ovulación (con hCG) tras el destete (se realizó una única inseminación cervical a las 37 ó 30 h de la inyección con hCG). El tratamiento de los espermatozoides de verraco con 1 mg CLC/120 x 106 de espermatozoides, amplió los límites de resistencia osmótica (tanto hipo- 50, 75 y 15

El objetivo de trabajo es el desarrollo de un protocolo para la adición de ciclodextrinas saturadas de colesterol (clc) al semen de caprino, con el fin de modificar la membrana plasmática de los espermatozoides y mejorar su calidad post-descongelación.