Productive performance, carcass and meat quality of intact and castrated gilts slaughtered at 106 or 122 kg BW

A total of 200 (Landrace3Large White dam3Pietrain3Large White sire) gilts of 5063 days of age (23.361.47 kg BW) were used to investigate the effects of castration (intact gilt, IG v. castrated gilt, CG) and slaughter weight (SW; 106 v. 122 kg BW) on productive performance, carcass and meat quality. Four treatments were arranged factorially and five replicates of 10 pigs each per treatment. Half of the gilts were ovariectomized at 58 days of age (8 days after the beginning of the trial at 29.861.64 kg BW), whereas the other half remained intact. The pigs were slaughtered at 106 or 122 kg BW. Meat samples were taken at Musculus longissimus thoracis at the level of the last rib and subcutaneous fat samples were taken at the tail insertion. For the entire experimental period, CG had higher ( P,0.05) BW gain and higher ( P,0.001) backfat and Musculus gluteus medius fat thickness than IG. However, IG had higher ( P,0.05) loin and trimmed primal cut yields than CG. Meat quality was similar for IG and CG but the proportion of linoleic acid in subcutaneous fat was higher ( P,0.001) for IG. Pigs slaughtered at 122 kg BW had higher ( P,0.001) feed intake and poorer feed efficiency than pigs slaughtered at 106 kg BW. An increase in SW improved ( P,0.001) carcass yield but decreased ( P,0.05) trimmed primal cut yield. Meat from pigs slaughtered at the heavier BW was redder (a*; P,0.001) and had more ( P,0.01) intramuscular fat and less thawing ( P,0.05) and cooking ( P,0.10) loss than meat from pigs slaughtered at the lighter BW. In addition, pigs slaughtered at 122 kg BW had less ( P,0.01) linoleic acid content in subcutaneous fat than pigs slaughtered at 106 kg BW. Castration of gilts and slaughtering at heavier BW are useful practices for the production of heavy pigs destined to the dry-cured industry in which a certain amount of fat in the carcass is required. In contrast, when the carcasses are destined to fresh meat production, IG slaughtered at 106 kg BW is a more efficient alternative.

Influence of castration on growth performance and carcass and meat quality of heavy female pigs

The main objetive of this Doctoral Thesis was to study the influence of female castration and pig sex on growth performance and carcass and meat quality of white pigs slaughtered at different final weights. Three experiments (Exp.) were conducted. In Exp. 1, a total of 200 (Landrace * Large White dam x Pietrain * Large White sire) gilts of 50 ± 3 days of age (23.3 ± 1.47 kg BW) was used to investigate the effects of castration (intact females, IF vs. castrated feamles, CF) and slaughter weight (106 vs. 122 kg BW) on productive performance and carcass and meat quality. There were four experimental treatments arranged as a 2 x 2 factorial and 5 replicates of 10 pigs each per treatment. Half of the gilts were ovariectomized at 58 d of age (8 days after the beginning of the trial; 29.8 ± 1.64 kg BW) whereas the other half remained intact. Meat samples were taken at m. Longissimus thoracis at the level of the last rib and subcutaneous fat samples were taken at the tail insertion. For the entire experiment period, CF had higher BW gain (P<0.05) and backfat and m. Gluteus medius (GM) fat thickness (P<0.001) than IF. However, IF had higher loin and trimmed primal cut yields (P<0.05) than CF. Meat quality was similar for IF and CF but the proportion of linoleic acid in subcutaneous fat was higher (P<0.001) for IF. Pigs slaughtered at 122 kg BW had higher (P<0.001) feed intake and poorer feed efficiency than pigs slaughtered at 106 kg BW. An increase in slaughter weight (SW) improved (P<0.001) carcass yield but decreased (P<0.05) trimmed primal cut yield. Meat from females slaughtered at the heavier BW was redder (a*; P<0.001) and had more (P<0.01) intramuscular fat and less thawing (P<0.05) and cooking (P<0.10) loss than meat from females slaughtered at the lighter BW. Also, females slaughtered at 122 kg BW had less (P<0.01) linoleic acid content in the subcutaneous fat than pigs slaughtered at 106 kg BW. Castration of gilts and slaughtering at heavier BW might be useful practices for the production of heavy pigs destined to the dry cured industry in which a certain amount of fat in the carcass is required. In contrast, when the carcasses are destined to fresh meat production, IF slaughtered at 106 kg BW are a more efficient alternative. In Exp. 2, crossbred pigs (n=240) from Pietrain*Large White sires mated to Landrace*Large White dams with an average of 100 d of age (60.5 ± 2.3 kg) were used to investigate the effects of gender and slaughter weight (SW) on growth performance and carcass and meat quality characteristics. There were 6 treatments arranged factorially with 3 genders (IF vs. CF vs.castrated males, CM) and 2 SW (114 vs. 122 kg BW). Each of the 6 combinations of treatments was replicated 4 times and the experimental unit was a pen with 10 pigs. Castrated males and CF ate more feed, grew faster and had more carcass backfat depth and fat thickness at the GM muscle, but lower loin yield than IF (P<0.05). In addition, CF and CM had more intramuscular fat (P<0.05) and less linoleic acid content in the subcutaneous fat (P<0.01) than IF. Pigs slaughtered at 122 kg BW had lower ADG (P<0.05), poor gain-to-feed ratio (P<0.05), and more GM fat than pigs slaughtered at 114 kg BW (P < 0.05). It is concluded that CF and CM had similar productive performance and meat quality characteristics when slaughtered at the same age, and that the castration of females improved daily gains and increased weight and fat content of primal cuts with respect to IF. Therefore, castration of females is recommended in pigs destined to the dry-cured industry because of the beneficial effects on the quality of the primal cuts. In Exp. 3, the effects of gender and castration of females (IF vs. CF vs. CM) on performance and carcass and meat quality were studied in crossbred pigs (Landrace x Large White dams x Duroc sires) slaughtered at 119.2 (trial 1) or 131.6 (trial 2) kg BW. Intact females had better feed conversion and less carcass fat than CF and CM. Trimmed shoulder yield was higher for CM than for CF with IF being intermediate. Primal cut yield and meat quality, however were similar for all treatments. Proportion of linoleic acid in backfat was lower for CF than for IF or CM, and the differences were significant in pigs slaughtered witn 131.6 kg BW. The higher fat content and the fatty acid profile favour the use of CF and CM over IF for the production of heavy pigs destined to the dry-cured industry.

Validation and aplication of Bioelectrical Impedance (BIA) method to determine body and carcass composition and nutrient retention of growing rabbits = Validación y aplicación de la técnica de la Impedancia Bioeléctrica (BIA) para determinar la composición corporal, de la canal y la retención de nutrientes de conejos en crecimiento

Los objetivos de esta tesis fueron 1) obtener y validar ecuaciones de predicción para determinar in vivo la composición corporal y de la canal de conejos en crecimiento de 25 a 77 días de vida utilizando la técnica de la Impedancia Bioeléctrica (BIA), y 2) evaluar su aplicación para determinar diferencias en la composición corporal y de la canal, así como la retención de nutrientes de animales alimentados con diferentes fuentes y niveles de grasa. El primer estudio se realizó para determinar y después validar, usando datos independientes, las ecuaciones de predicción obtenidas para determinar in vivo la composición corporal de los conejos en crecimiento. Se utilizaron 150 conejos a 5 edades distintas (25, 35, 49, 63 y 77 días de vida), con un rango de pesos entre 231 y 3138 g. Para determinar los valores de resistencia (Rs,) and reactancia (Xc,) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) de cada animal. En cada edad, los animales fueron molidos y congelados (-20 ºC) para su posterior análisis químico (MS, grasa, proteína, cenizas y EB). El contenido en grasa y energía de los animales se incrementó, mientras que los contenidos en proteína, cenizas y agua de los animales disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm y 10.8 ± 3.1 cm. Se realizó un análisis de regresión lineal múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) tuvieron un coeficiente de determinación de (R2) de 0.99, 0.99, 0.97, 0.98 y 0.99, y los errores medios de predicción relativos (EMPR) fueron: 2.79, 6.15, 24.3, 15.2 y 10.6%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.85 and 2.30%, respectivamente. Al predecir los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), se obtuvieron valores de 0.79, 0.83, 0.71 y 0.86 para R2, y 5.04, 18.9, 12.0 y 3.19% para EMPR. La reactancia estuvo negativamente correlacionada con el contenido en agua, cenizas y PB (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) y positivamente correlacionada con la grasa y la energía (r = 0.23 y r = 0.24; P < 0.01). Sin embargo, Rs estuvo positivamente correlacionada con el agua, las cenizas y la PB (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) y negativamente con la grasa y la energía (r = -0.36 y r = -0.35; P < 0.0001). Igualmente la edad estuvo negativamente correlacionada con el contenido en agua, cenizas y proteína (r = -0.79; r = -0.68 y r = -0.80; P < 0.0001) y positivamente con la grasa y la energía (r = 0.78 y r = 0.81; P < 0.0001). Se puede concluir que el método BIA es una técnica buena y no invasiva para estimar in vivo la composición corporal de conejos en crecimiento de 25 a 77 días de vida. El objetivo del segundo estudio fue determinar y validar con datos independientes las ecuaciones de predicción obtenidas para estimar in vivo la composición de la canal eviscerada mediante el uso de BIA en un grupo de conejos de 25 a 77 días, así como testar su aplicación para predecir la retención de nutrientes y calcular las eficacias de retención de la energía y del nitrógeno. Se utilizaron 75 conejos agrupados en 5 edades (25, 35, 49, 63 y 77 días de vida) con unos pesos que variaron entre 196 y 3260 g. Para determinar los valores de resistencia (Rs, ) y reactancia (Xc, ) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) del cada animal. En cada edad, los animales fueron aturdidos y desangrados. Su piel, vísceras y contenido digestivo fueron retirados, y la canal oreada fue pesada y molida para posteriores análisis (MS, grasa, PB, cenizas y EB). Los contenidos en energía y grasa aumentaron mientras que los de agua, cenizas y proteína disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. Se realizó un análisis de regresión linear múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Los coeficientes de determinación (R2) de las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) fueron: 0.99, 0.99, 0.95, 0.96 y 0.98, mientras que los errores medios de predicción relativos (EMPR) fueron: 4.20, 5.48, 21.9, 9.10 y 6.77%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.79 y 1.62%, respectivamente. Cuando se realizó la predicción de los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), los valores de R2 fueron 0.68, 0.76, 0.66 and 0.82, y los de RMPE: 3.22, 10.5, 5.82 and 2.54%, respectivamente. La reactancia estuvo directamente correlacionada con el contenido en grasa (r = 0.24, P < 0.05), mientras que la resistencia guardó una correlación positiva con los contenidos en agua, cenizas y proteína (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) y negativa con la grasa y la energía (r = -0.44 y r = -0.55; P < 0.001). Igualmente la edad estuvo negativamente correlacionada con los contenidos en agua, cenizas y PB (r = -0.94; r = -0.85 y r = -0.75; P < 0.0001) y positivamente con la grasa y la energía (r = 0.89 y r = 0.90; P < 0.0001). Se estudió la eficacia global de retención de la energía (ERE) y del nitrógeno (ERN) durante todo el periodo de cebo (35-63 d), Los valores de ERE fueron 20.4±7.29%, 21.0±4.18% and 20.8±2.79% en los periodos 35 a 49, 49 a 63 y 35 a 63 d, respectivamente. ERN fue 46.9±11.7%, 34.5±7.32% y 39.1±3.23% para los mismos periodos. La energía fue retenida en los tejidos para crecimiento con una eficiencia del 52.5% y la eficiencia de retención de la energía como proteína y grasa fue de 33.3 y 69.9% respectivamente. La eficiencia de utilización del nitrógeno para crecimiento fue cercana al 77%. Este trabajo muestra como el método BIA es técnica buena y no invasiva para determinar in vivo la composición de la canal y la retención de nutrientes en conejos en crecimiento de 25 a 77 días de vida. En el tercer estudio, se llevaron a cabo dos experimentos con el fin de investigar los efectos del nivel de inclusión y de la fuente de grasa, sobre los rendimientos productivos, la mortalidad, la retención de nutrientes y la composición corporal total y de la canal eviscerada de conejos en crecimiento de 34 a 63 d de vida. En el Exp. 1 se formularon 3 dietas con un diseño experimental factorial 3 x 2 con el tipo de grasa utilizada: Aceite de Soja (SBO), Lecitinas de Soja (SLO) y Manteca (L) y el nivel de inclusión (1.5 y 4%) como factores principales. El Exp. 2 también fue diseñado con una estructura factorial 3 x 2, pero usando SBO, Aceite de Pescado (FO) y Aceite de Palmiste como fuentes de grasa, incluidas a los mismos niveles que en el Exp. 1. En ambos experimentos 180 animales fueron alojados en jaulas individuales (n=30) y 600 en jaulas colectivas en grupos de 5 animales (n=20). Los animales alimentados con un 4% de grasa añadida tuvieron unos consumos diarios y unos índices de conversión más bajos que aquellos alimentados con las dietas con un 1.5% de grasa. En los animales alojados en colectivo del Exp. 1, el consumo fue un 4.8% más alto en los que consumieron las dietas que contenían manteca que en los animales alimentados con las dietas SBO (P = 0.036). La inclusión de manteca tendió a reducir la mortalidad (P = 0.067) en torno al 60% y al 25% con respecto a las dietas con SBO y SLO, respectivamente. La mortalidad aumentó con el nivel máximo de inclusión de SLO (14% vs. 1%, P < 0.01), sin observarse un efecto negativo sobre la mortalidad con el nivel más alto de inclusión de las demás fuentes de grasa utilizadas. En los animales alojados colectivo del Exp. 2 se encontró una disminución del consumo (11%), peso vivo a 63 d (4.8%) y de la ganancia diaria de peso (7.8%) con la inclusión de aceite de pescado con respecto a otras dietas (P < 0.01). Los dos últimos parámetros se vieron especialmente más reducidos cuando en las dietas se incluyó el nivel más alto de FO (5.6 y 9.5%, respectivamente, (P < 0.01)). Los animales alojados individualmente mostraron unos resultados productivos muy similares. La inclusión de aceite pescado tendió (P = 0.078) a aumentar la mortalidad (13.2%) con respecto al aceite de palmiste (6.45%), siendo intermedia para las dietas que contenían SBO (8.10%). La fuente o el nivel de grasa no afectaron la composición corporal total o de la canal eviscerada de los animales. Un incremento en el nivel de grasa dio lugar a una disminución de la ingesta de nitrógeno digestible (DNi) (1.83 vs. 1.92 g/d; P = 0.068 en Exp. 1 y 1.79 vs. 1.95 g/d; P = 0.014 en Exp. 2). Debido a que el nitrógeno retenido (NR) en la canal fue similar para ambos niveles (0.68 g/d (Exp. 1) y 0.71 g/d (Exp. 2)), la eficacia total de retención del nitrógeno (ERN) aumentó con el nivel máximo de inclusión de grasa, pero de forma significativa únicamente en el Exp. 1 (34.9 vs. 37.8%; P < 0.0001), mientras que en el Exp. 2 se encontró una tendencia (36.2 vs. 38.0% en Exp. 2; P < 0.064). Como consecuencia, la excreción de nitrógeno en heces fue menor en los animales alimentados con el nivel más alto de grasa (0.782 vs. 0.868 g/d; P = 0.0001 en Exp. 1, y 0.745 vs. 0.865 g/d; P < 0.0001 en Exp.2) al igual que el nitrógeno excretado en orina (0.702 vs. 0.822 g/d; P < 0.0001 en Exp. 1 y 0.694 vs. 0.7999 g/d; P = 0.014 en Exp.2). Aunque no hubo diferencias en la eficacia total de retención de la energía (ERE), la energía excretada en heces disminuyó al aumentar el nivel de inclusión de grasa (142 vs. 156 Kcal/d; P = 0.0004 en Exp. 1 y 144 vs. 154 g/d; P = 0.050 en Exp. 2). Sin embargo, la energía excretada como orina y en forma de calor fue mayor en el los animales del Exp. 1 alimentados con el nivel más alto de grasa (216 vs. 204 Kcal/d; P < 0.017). Se puede concluir que la manteca y el aceite de palmiste pueden ser considerados como fuentes alternativas al aceite de soja debido a la reducción de la mortalidad, sin efectos negativos sobre los rendimientos productivos o la retención de nutrientes. La inclusión de aceite de pescado empeoró los rendimientos productivos y la mortalidad durante el periodo de crecimiento. Un aumento en el nivel de grasa mejoró el índice de conversión y la eficacia total de retención de nitrógeno. ABSTRACT The aim of this Thesis is: 1) to obtain and validate prediction equations to determine in vivo whole body and carcass composition using the Bioelectrical Impedance (BIA) method in growing rabbits from 25 to 77 days of age, and 2) to study its application to determine differences on whole body and carcass chemical composition, and nutrient retention of animals fed different fat levels and sources. The first study was conducted to determine and later validate, by using independent data, the prediction equations obtained to assess in vivo the whole body composition of growing rabbits. One hundred and fifty rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) and weighing from 231 to 3138 g were used. A four terminal body composition analyser was used to obtain resistance (Rs, ) and reactance (Xc, ) values (Model BIA-101, RJL Systems, Detroit, MI USA). The distance between internal electrodes (D, cm), body length (L, cm) and live BW of each animal were also registered. At each selected age, animals were slaughtered, ground and frozen (-20 ºC) for later chemical analyses (DM, fat, CP, ash and GE). Fat and energy body content increased with the age, while protein, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm and 10.8 ± 3.1 cm. A multiple linear regression analysis was used to determine the prediction equations, using BW, L and Z data as independent variables. Equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content had, respectively, coefficient of determination (R2) values of 0.99, 0.99, 0.97, 0.98 and 0.99, and the relative mean prediction error (RMPE) was: 2.79, 6.15, 24.3, 15.2 and 10.6%, respectively. When water was expressed as percentage, the R2 and RMPE were 0.85 and 2.30%, respectively. When prediction of the content of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) was done, values of 0.79, 0.83, 0.71 and 0.86 for R2, and 5.04, 18.9, 12.0 and 3.19% for RMPE, respectively, were obtained. Reactance was negatively correlated with water, ash and CP content (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) and positively correlated with fat and GE (r = 0.23 and r = 0.24; P < 0.01). Otherwise, resistance was positively correlated with water, ash and CP (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) and negatively correlated with fat and energy (r = -0.36 and r = -0.35; P < 0.0001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.79; r = -0.68 and r = -0.80; P < 0.0001) and positively correlated with fat and energy (r = 0.78 and r = 0.81; P < 0.0001). It could be concluded that BIA is a non-invasive good method to estimate in vivo whole body composition of growing rabbits from 25 to 77 days of age. The aim of the second study was to determine and validate with independent data, the prediction equations obtained to estimate in vivo carcass composition of growing rabbits by using the results of carcass chemical composition and BIA values in a group of rabbits from 25 to 77 days. Also its potential application to predict nutrient retention and overall energy and nitrogen retention efficiencies was analysed. Seventy five rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) with weights ranging from 196 to 3260 g were used. A four terminal body composition analyser (Model BIA-101, RJL Systems, Detroit, MI USA) was used to obtain resistance (Rs, ) and reactance (Xc, ) values. The distance between internal electrodes (D, cm), body length (L, cm) and live weight (BW, g) were also registered. At each selected age, all the animals were stunned and bled. The skin, organs and digestive content were removed, and the chilled carcass were weighed and processed for chemical analyses (DM, fat, CP, ash and GE). Energy and fat increased with the age, while CP, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. A multiple linear regression analysis was done to determine the equations, using BW, L and Z data as parameters. Coefficient of determination (R2) of the equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content were: 0.99, 0.99, 0.95, 0.96 and 0.98, and relative mean prediction error (RMPE) were: 4.20, 5.48, 21.9, 9.10 and 6.77%, respectively. When water content was expressed as percentage, the R2 and RMPE were 0.79 and 1.62%, respectively. When prediction of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) content was done, R2 values were 0.68, 0.76, 0.66 and 0.82, and RMPE: 3.22, 10.5, 5.82 and 2.54%, respectively. Reactance was positively correlated with fat content (r = 0.24, P < 0.05) while resistance was positively correlated with water, ash and protein carcass content (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) and negatively correlated with fat and energy (r = -0.44 and r = -0.55; P < 0.001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.97, r = -0.95 and r = -0.89, P < 0.0001) and positively correlated with fat and GE (r = 0.95 and r = 0.97; P < 0.0001). In the whole growing period (35-63 d), overall energy retention efficiency (ERE) and nitrogen retention efficiency (NRE) were studied. The ERE values were 20.4±7.29%, 21.0±4.18% and 20.8±2.79%, from 35 to 49, 49 to 63 and from 35 to 63 d, respectively. NRE was 46.9±11.7%, 34.5±7.32% and 39.1±3.23% for the same periods. Energy was retained in body tissues for growth with an efficiency of approximately 52.5% and efficiency of the energy for protein and fat retention was 33.3 and 69.9%, respectively. Efficiency of utilization of nitrogen for growth was near to 77%. This work shows that BIA it’s a non-invasive and good method to estimate in vivo carcass composition and nutrient retention of growing rabbits from 25 to 77 days of age. In the third study, two experiments were conducted to investigate the effect of the fat addition and source, on performance, mortality, nutrient retention, and the whole body and carcass chemical composition of growing rabbits from 34 to 63 d. In Exp. 1 three diets were arranged in a 3 x 2 factorial structure with the source of fat: Soybean oil (SBO), Soya Lecithin Oil (SLO) and Lard (L) and the dietary fat inclusion level (1.5 and 4%) as the main factors. Exp. 2 had also arranged as a 3 x 2 factorial design, but using SBO, Fish Oil (FO) and Palmkernel Oil (PKO) as fat sources, and included at the same levels than in Exp. 1. In both experiments 180 animals were allocated in individual cages (n=30) and 600 in collectives cages, in groups of 5 animals (n=20). Animals fed with 4% dietary fat level showed lower DFI and FCR than those fed diets with 1.5%. In collective housing of Exp. 1, DFI was a 4.8% higher in animals fed with diets containing lard than SBO (P = 0.036), being intermediate for diet with SLO. Inclusion of lard also tended to reduce mortality (P = 0.067) around 60% and 25% with respect SBO and SLO diets, respectively. Mortality increased with the greatest level of soya lecithin (14% vs. 1%, P < 0.01). In Exp. 2 a decrease of DFI (11%), BW at 63 d (4.8%) and DWG (7.8%) were observed with the inclusion of fish oil with respect the other two diets (P < 0.01). These last two traits impaired with the highest level of fish oil (5.6 and 9.5%, respectively, (P < 0.01)). Animals housed individually showed similar performance results. The inclusion of fish oil also tended to increase (P = 0.078) mortality (13.2%) with respect palmkernel oil (6.45%), being mortality of SBO intermediate (8.10%). Fat source and level did not affect the whole body or carcass chemical composition. An increase of the fat sources addition led to a decrease of the digestible nitrogen intake (DNi) (1.83 vs. 1.92 g/d; P = 0.068 in Exp. 1 and 1.79 vs. 1.95 g/d; P = 0.014 in Exp. 2). As the nitrogen retained (NR) in the carcass was similar for both fat levels (0.68 g/d (Exp. 1) and 0.71 g/d (Exp. 2)), the overall efficiency of N retention (NRE) increased with the highest level of fat, but only reached significant level in Exp. 1 (34.9 vs. 37.8%; P < 0.0001), while in Exp. 2 a tendency was found (36.2 vs. 38.0% in Exp. 2; P < 0.064). Consequently, nitrogen excretion in faeces was lower in animals fed with the highest level of fat (0.782 vs. 0.868 g/d; P = 0.0001 in Exp. 1, and 0.745 vs. 0.865 g/d; P < 0.0001 in Exp.2). The same effect was observed with the nitrogen excreted as urine (0.702 vs. 0.822 g/d; P < 0.0001 in Exp. 1 and 0.694 vs. 0.7999 g/d; P = 0.014 in Exp.2). Although there were not differences in ERE, the energy excreted in faeces decreased as fat level increased (142 vs. 156 Kcal/d; P = 0.0004 in Exp. 1 and 144 vs. 154 g/d; P = 0.050 in Exp. 2). In Exp. 1 the energy excreted as urine and heat production was significantly higher when animals were fed with the highest level of dietary fat (216 vs. 204 Kcal/d; P < 0.017). It can be concluded that lard and palmkernel oil can be considered as alternative sources to soybean oil due to the reduction of the mortality, without negative effects on performances or nutrient retention. Inclusion of fish impaired animals´ productivity and mortality. An increase of the dietary fat level improved FCR and overall protein efficiency retention.

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration.

Characterization of two patched receptors for the vertebrate hedgehog protein family

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33–34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

How vertebrate and invertebrate visual pigments differ in their mechanism of photoactivation

In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the “counterion” is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation—that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.

Small molecule developmental screens reveal the logic and timing of vertebrate development

Much has been learned about vertebrate development by random mutagenesis followed by phenotypic screening and by targeted gene disruption followed by phenotypic analysis in model organisms. Because the timing of many developmental events is critical, it would be useful to have temporal control over modulation of gene function, a luxury frequently not possible with genetic mutants. Here, we demonstrate that small molecules capable of conditional gene product modulation can be identified through developmental screens in zebrafish. We have identified several small molecules that specifically modulate various aspects of vertebrate ontogeny, including development of the central nervous system, the cardiovascular system, the neural crest, and the ear. Several of the small molecules identified allowed us to dissect the logic of melanocyte and otolith development and to identify critical periods for these events. Small molecules identified in this way offer potential to dissect further these and other developmental processes and to identify novel genes involved in vertebrate development.

Morphological castes in a vertebrate

Morphological specialization for a specific role has, until now, been assumed to be restricted to social invertebrates. Herein we show that complete physical dimorphism has evolved between reproductives and helpers in the eusocial naked mole-rat. Dimorphism is a consequence of the lumbar vertebrae lengthening after the onset of reproduction in females. This is the only known example of morphological castes in a vertebrate and is distinct from continuous size variation between breeders and helpers in other species of cooperatively breeding vertebrates. The evolution of castes in a mammal and insects represents a striking example of convergent evolution for enhanced fecundity in societies characterized by high reproductive skew. Similarities in the selective environment between naked mole-rats and eusocial insect species highlight the selective conditions under which queen/worker castes are predicted to evolve in animal societies.