Proceedings Undergraduate Research Experiences
Transcripción
Proceedings Undergraduate Research Experiences
Proceedings Undergraduate Research Experiences 2015, Vol. 1:1 Resident Instruction for Insular Areas Educational Grant Program 2015 Resident Instruction Grants Program for Institutions of Higher Education in Insular Areas Undergraduate Research Experiences Participating Students and Faculty Student Mentor Area of Expertise Andréa Paola Ríos [email protected] Abner A. Rodríguez [email protected] Ruminant Nutrition Forage Conservation Natalie Vázquez [email protected] Esbal Jiménez [email protected] Animal Reproduction Miltón González Serrano [email protected] Leyda Ponce de León [email protected] Dairy Products Camille Cordero [email protected] Jaime Curbelo [email protected] Dairy Cattle Mastitis Nicolle Irizarry Larriuz [email protected] Paul F. Randel [email protected] Animal Nutrition Abdiel Santana Rivera [email protected] David Sotomayor [email protected] Soil and Water Quality Soil Fertility Jennifer Rivera San Antonio [email protected] Roberto Vargas [email protected] Nematology Mibet Cordero [email protected] Maria del Carmen Librán [email protected] Horticulture Alexandra Ramírez Irizarry [email protected] Pablo Morales Payan [email protected] Organic Agriculture Veronica Rivera [email protected] Lydia Rivera [email protected] Tropical Plant Pathology/Mycology Abner A. Rodríguez Carías, Ph.D. Professor Department of Animal Science Project Coordinator Table of Content Intake, growth performance, and carcass yield of meat-type rabbits fed with different levels of corn stalk silage. 1 Andrea P. Ríos, Abner A. Rodríguez and Luis C. Solórzano Expression of prolactin receptor in the skin of dry Holstein cows with phenotypic differences in hair coat. 6 Natalie M. Vázquez and Esbal Jiménez Use of exopolysaccharides producing lactic acid bacteria in the manufacture of low fat Gouda cheese. Milton S. González and Leyda Ponce de León 10 The effect of scrotal temperature on semen quality parameters in Holstein Friesian and Jersey bulls. 16 Camille Cordero-Aponte and Jaime E. Curbelo-Rodríguez The retained placenta malady in a Puerto Rican dairy cattle herd: prevalence, treatment and effects on animal performance. 23 Nicole M. Irizarry, Paul F. Randel, Héctor Santana and Ricardo Montañez Soil nitrogen mineralization in soils cropped to maize (Zea Mays) of the southern semiarid zone of Puerto Rico. 29 Abdiel Santana, Johannies Rivera-Zayas and David Sotomayor-Ramírez Comparative Study of Organic and Conventional Farms in Puerto Rico Using Nematode Communities as Environmental Indicators. 37 Jennifer M. Rivera San Antonio and Roberto Vargas Growth responses of lettuce plants (Lactuca sativa L., cv. “Black Seeded Simpson”) grown under a hydroponic system (NFT) receiving inorganic and organic nutrient solution. 42 Mibet M. Cordero Ruiz and María del Carmen Librán Effects of Exogenous Biostimulants on Tahiti Lime (Citrus latifolia) Trees Affected by the Citrus Greening Disease in Lajas, Puerto Rico. 51 Alexandra I. Ramírez-Irizarry and J. Pablo Morales-Payan Rhytismatales and other pathogenic fungal species of native trees of Puerto Rico Verónica M. Rivera Vega and Lydia. I. Rivera Vargas 58 Intake, growth performance, and carcass yield of meat-type rabbits fed with different levels of corn stalk silage Andrea P. Ríos, Abner A. Rodríguez and Luis C. Solórzano Department of Animal Science Abstract Among the most important problems facing rabbit producers in Puerto Rico is the high cost of commercial feed (CF), therefore, alternatives to its use need to be evaluate. Rabbits are nonruminant herbivores capable of degrading fiber, thus inclusion of fermented forages might represent a useful alternative. An experiment was conducted to evaluate the inclusion of corn stalk silage (CS; 28% DM) in diets for growing rabbits based on voluntary intake, growth performance, and carcass yield. Thirty New Zealand white rabbits (averaging 5 weeks of age and 700g live weight) were distributed by initial weight into 30 elevated cages and randomly assigned to three treatments; control (T1, 100% CF), and inclusion of CS at 25% (T2) and 50% (T3) of the total dry matter offered. Diets were offered at 5% of rabbit body weigh daily on a dry matter basis during 59 days. Data collected on feed intake, weight gain, feed conversion, and carcass dressing percentage were statistically analyzed using a completely randomized design with 10 replicates per treatment and Bonferroni-test for mean separation. Total intake was higher (P<0.05) in rabbits fed the 100% CF diet (59.03g/d), than in those offered 50% CS (40.74g/d), but not significantly different from those fed 25% CS (49.53g/d). Control rabbits showed higher (P<0.05) daily gain (T1=17.58 g, T2=12.46 g, T3=6.60 g) and lower feed conversion ratio than animals that received CS in the diet (T1=3.38, T2=4.01, T3=6.17). Carcass yield percentage was lower (P<0.05) in animals fed diets containing 50% CS than in those fed 0 and 25% CS. In summary, corn stalk silage at 25% and 50% inclusion rates can be used in growing rabbit diets but with resulting lower productive performance than that obtained by animals fed 100% CF. Inclusion of CS in the diet at the 25% level did not affect rabbit carcass yield. Keywords: Rabbits, Corn Silage, Performance Resumen Entre los principales problemas que enfrentan los productores de conejos en Puerto Rico se encuentra el alto costo del alimento concentrado (AC), por esta razón las alternativas de alimentación deben ser evaluadas. Los conejos son herbívoros no rumiantes con capacidad de degradar fibra, por lo que la inclusión de forrajes conservados en su alimentación podría representar una alternativa útil. Se realizó un experimento para evaluar la inclusión de ensilaje de materia vegetativa de maíz (EM; 28% MS) en dietas para conejos destinados a la producción de carne, basado en los criterios de ingesta voluntaria, ganancia en peso y rendimiento de la canal. Treinta conejos de la raza Nueva Zelandia blanco (promedio de 5 semanas de edad y 700 g de peso vivo) fueron distribuidos, según su peso inicial, en 30 jaulas elevadas y asignados aleatoriamente a tres tratamientos; control (T1, 100% AC), y la inclusión de EM a 25 (T2) y 50% (T3) del total ofrecido en materia seca. Las dietas se ofrecieron diariamente al 5% del peso vivo del animal en base seca durante 59 días. Se recolectaron datos de consumo de alimento, ganancia o pérdida de peso, conversión alimenticia y rendimiento de la canal. Los datos se analizaron según un diseño completamente al azar con 10 repeticiones por tratamiento. La prueba de Bonferroni se utilizó para la separación de medias. El consumo total fue mayor (P <0.05) en los 1 conejos alimentados con la dieta AC 100% (59.03 g/d), que en los que se les ofrecieron 50% EM (40.74 g/d) o 25% EM (49.53 g/d). Los conejos de control presentaron mayor (P <0.05) ganancia diaria en peso (T1= 17,58 g, T2= 12,46 g, T3= 6,60 g) y una menor (P<0.05) conversión alimenticia que los animales que recibieron EM en la dieta (T1= 3.38, T2= 4.01, T3= 6.17). El porcentaje de rendimiento de la canal fue menor (P <0,05) en los animales alimentados con dietas que contenían 50% EM que en los alimentados con 0 y 25% EM. En resumen, el incluir ensilaje de maíz en 25 y 50% de la materia seca dietética puede ser utilizado para alimentar conejos, pero este presenta un menor rendimiento productivo comparado con el obtenido por los animales alimentados con 100% AC. Inclusión de EM en la dieta a un nivel de 25% no afectó el rendimiento de la canal. Palabras Claves: Conejos, Ensilaje de Maíz, Rendimiento Introduction The last agricultural census in Puerto Rico reports that commercial rabbit farms and animal inventory have been declining. In 2007, there were 119 commercial farms and 38,519 rabbits, numbers that decreased in 2012 to 98 farms and 33,982 animals (USDA/NASS, 2014). Feeding cost is among the most important problems that affect local rabbit producers. Currently, rabbit diets are based exclusively of commercial feeds, which are disadvantageous due to: high price, low energy density, and inadequate fiber content. Rabbits are non–ruminant herbivores that practice hindgut fermentation and are capable of degrading fiber. Thus, they can utilize forages and fibrous by – product feeds as a major dietary component (Tonson et al., 1999). Inclusion of fermented forages in rabbit diets could represent a useful option to reduce feed costs for commercial rabbit producers. Corn silage might be a candidate for this purpose because of its high yield, good nutritive value and easy handling. This study was designed, to evaluate the inclusion of corn stalk silage as an integral part of the diet for growing meat–type rabbits based on the criteria intake, growth performance and carcass yield. Material and Methods The study was conducted in a pavilion specialized for rabbit production located in the Alzamora Farm at the University of Puerto Rico, Mayagüez Campus (Figure 1). Thirty New Zealand White rabbits (averaging 5 weeks of age and 700g live weight) were obtained from the UPRM Agricultural Experiment Station Small Animal Research Farm in Lajas. Animals were distributed by initial weight into thirty elevated cages (Figure 2) and randomly assigned to three treatments; control (T1, 100% CF), and addition of corn stalk silage (CS) at 25% (T2) and 50% (T3) levels. The silage was prepared from a Mayorbela variety, harvested at 90 d. of growth forage. The was chopped in to pieces of 2 -3 cm length and ensiled in 8-gal plastic silos for a minimum of 21 d. Diets were offered daily at 5% of rabbit live weight on dry matter a basis during 59 days (Figure 3). Nutrient composition of both feedstuffs was determined using standard procedures (AOAC, 1990; Van Soest et al., 1991), while pH and concentrations of fermentation products of CS were determined in a commercial laboratory. The offered feed and orts were weighted daily and total intake was calculated. Rabbit body weight was recorded weekly (Figure 4) and used to determine total and average daily gain and feed to gain conversation ratio. After 60 days of experimentation the animals were sacrificed in the slaughterhouse of the Small Animal facilities in Lajas (Figure 5). 2 Figure 1. Pavilion for Rabbit Production Figure 2. Elevated Rabbit Cages Figure 3. Diet Daily Offering At slaughter hot carcass weight was recorded (Figure 6) to determine carcass-dressing percentage. Liver, kidney and stomach weighs were also recorded and expressed as percentage of final weight (Figure 7). Data collected on feed intake, weight gain, feed conversion, carcass dressing percentage, and organ weights were statically analyzed using a completely randomized design with 10 replicates per treatment and Bonferroni-test for mean separation. Figure 4. Rabbit Body Weight Measurement Figure 5. Slaughtered Animals Figure 6. Hot Carcass Weight Measurement Figure 7. Organ Weight Measurement 3 Results and Discussion The nutrient profile of the CF utilized in this experiment is in agreement with the manufacture’s label, while the CS had a chemical composition and ensiling characteristics typical of those observed for corn stalks ensiled in a tropical environment (Table 1). Obviously the starch content of only 0.4% is much lower than that of CS including the grain. Total intake was higher (P<0.05) in rabbits fed the 100% CF diet (59.0g/d), than in those offered 50% CS (40.7g/d), but not significantly different from those fed 25% CS (49.53g/d). Control rabbits showed higher (P<0.05) daily gain (T1=17.5 g, T2=12.4 g, T3=6.60 g) and more efficient feed conversion than animals receiving either level of CS in the diet (T1= 3.38, T2= 4.01, T3= 6.1). Carcass yield percentage was lower (P<0.05) in animals fed the diet containing 50% CS than in those fed 0 and 25% CS. Similar organ weights as a percentage of final rabbit weight were observed in the three treatments. In this experiment inclusion of both levels of CS resulted in lower intake and productive performance than that obtained by rabbits fed 100% CF. The CS - containing diets had lower crude protein and starch contents than the 100% CF, which likely affected animal performance. Therefore, to accomplish desirable growing rates in rabbits fed CS, ensiling of vegetative material with higher starch content in combination with the use of protein supplementation would seem to be necessary. Under the conditions of the present experiment, inclusion of CS in the diet at the 25% level did not significantly affect rabbit carcass yield, but lower yield was observed when CS was included at 50%, which is probably a reflection of lower nutrient density of the diet. Table 1. Chemical composition and fermentation profile of commercial feed and corn silage Chemical Composition (%) Item CF CS CS DM 93.1 26.4 pH 3.92 OM1 93.5 97.6 Lactic Acid 6.611 ASH1 6.5 2.4 CP1 Starch1 NDF1 ADF1 Lignin1 17.6 17.8 37.1 22.1 6.0 9.5 0.4 63.5 41.1 5.9 Ensiling Characteristics (%) Acetic N-H3/N Acid L/A Butyric Acid Propionic Acid Total 1.961 3.371,2 .171 .041 10.1 1 Dry matter basis, 2Lactic acid/acetic acid ratio Conclusion Corn silage at 25 and 50% inclusion rates can be use in growing rabbit diets but with lower productive performance than that obtained by animals fed 100% CF especially at the higher level of inclusion. Inclusion of CS in the diet at the 25% level resulted in only a small not significant effect on rabbit carcass yield. 4 Table 2. Intake, performance, and carcass yield of meat – type rabbits fed with different proportions of commercial feed and corn stalk silage Component Intake (g/d) CC CS Total 100:0 Proportion CF:CS 75:25 59.03a ---59.03a 38.9b 10.5b 48.5ab 22.4c 18.3a 40.7b .01 .01 .01 Body weight (g) Initial Final 695.0 1732.1a 699.4 1434.7b 688.1 1077.6c .97 .01 Gain (g) Daily Total 17.5a 1037.3a 12.4b 735.27b 6.6c 389.4c .01 .01 Feed Conversion Ratio 3.38b 4.01b 6.17a .01 Hot carcass yield (%) 49.2a 47.7a 43.8b .01 Organ weigh as % final weight Liver Stomach Kidney 5.91 14.0 1.3 5.93 13.2 1.3 5.02 12.4 1.3 .12 .39 .93 50:50 P a,b Means with unlike superscripts in the same row differ p<0.05 Cited Literature AOAC. 1990. Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists. Arlington, VA. Tonson, M.A.; S.A. Abd El-Lateif and M.R.M. Ibrahim 1999. Growth performance of NZW rabbits fed diets containing cassava leaves and stems meal as a substitution of clover hay meal. Egyptian J.Nutr. and Feeds, 2: 541. USDA/NASS. National Agricultural Statistical Services. Nass.usda.gov. 2014 http://agcensus.usda.gov/Publications/2012/Full_Report/Puerto_Rico/st72_1_014.pdf Van Soest, P. J., Robertson J. B. and Lewis, B. A. 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccarides in relation to animal nutrition. J. Dairy Sci. 74: 3583 – 3597. 5 Expression of prolactin receptor in the skin of dry Holstein cows with phenotypic differences in hair coat Natalie M. Vázquez and Esbal Jiménez Department of Animal Science Abstract The development of technologies to ameliorate the effects of heat stress in dairy cattle to maintain high milk production has been a long-term goal of researchers. These technologies in many cases augment the costs of producing milk. Recently, phenotypic differences (animals with shorter hair) were discovered in dairy cattle which may be an adaptation to heat stress. Increased secretion of prolactin has been observed in animals under heat stress. This hormone, which acts by activating the prolactin receptor (PRLR), also has been implicated with the growth of hair follicles in many mammals. Consequently, a study was performed to investigate if differences exist in the expression of PRLR between short-haired (SH) and normal-haired (NH) cows. Skin biopsies were obtained from 12 Holstein non-pregnant, dry cows; six classified as SH and six NH. RNA was extracted from the skin biopsies for cDNA synthesis. Further amplification and determination of relative expression of PRLR was determined by using specific primers and Real time PCR analysis. The relative expression of PRLR in SH and NH cows was similar (P=0.17). Numerically, the relative expression of PRLR in SH cows was 1.09 times greater than NH cows. Further investigations should test if structural differences exist in PRLR of SH and NH cows, which might increase or decrease the actions of prolactin in the skin of these animals. Keywords: short hair, prolactin receptor expression, dairy cattle Resumen El desarrollo de tecnologías para disminuir los efectos del estrés por calor en el ganado lechero, para mantener una alta producción de leche, ha sido un objetivo por muchos años de los investigadores. El problema con estas tecnologías es que aumentan los costos de producción de leche. Recientemente, diferencias fenotípicas (animales con pelo más corto) en la capa del pelo fueron descubiertos en el ganado lechero, lo que puede ser una adaptación al estrés por calor. Se ha observado que la secreción de prolactina aumenta en animales bajo estrés por calor. Esta hormona, que actúa mediante la activación del receptor de prolactina (PRLR), también ha sido implicada con el crecimiento de los folículos pilosos en muchos mamíferos. Por lo tanto, se realizó un estudio para investigar si existen diferencias en la expresión de PRLR entre vacas con pelo corto (SH) y pelo normal (NH). Se obtuvieron biopsias de la piel de 12 vacas Holstein no preñadas y horras; seis clasificadas como SH y seis NH. Se extrajo ARN de las biopsias de piel para la síntesis de cDNA. Luego se amplificó y determinó la expresión relativa de PRLR mediante el uso de cebadores específicos y análisis de PCR en tiempo real. La expresión relativa de PRLR en vacas SH y NH fue similar (P = 0.17). Numéricamente, la expresión relativa de PRLR en vacas SH fue 1.09 veces mayor que las vacas NH. Estudios adicionales deben realizarse para investigar si existen diferencias estructurales en PRLR de vacas SH y NH, que puedan aumentar o disminuir la acción de prolactina en la piel de estos animales. Palabras Claves: pelo corto, la expresión del receptor de prolactina, ganado lechero 6 Introduction The dairy industry suffers an economic crisis due to increases in production costs such as food, water, electricity and labor. It is therefore very important to find methods to improve productivity efficiency in dairy farms. Phenotypic differences in hair coat were discovered in dairy cattle possibly as an adaptation to heat stress. Specifically, the slick hair (SH) cow is the animal with shorter, thinner, and shinier hair (Olson, 2006). There have been several studies that compare milk production between both groups that found that SH cows have a higher milk production, lower vaginal temperature and lower respiration rate compared to NH (Olson, 2006). Prolactin (PRL), known primarily as a lactogenic hormone, has been observed to increased in animals under heat stress. This hormones, which acts by activating the prolactin receptor (PRLR) has also been implicated in the process of angiogenesis, osmoregulation, immune response, and hair growth and development; it also has a direct relationship to the environment’s temperature (Alamer, 2011). The PRLR are found mainly in the hair follicle, skin and around the sweat glands (Nixon et al., 2002). SH cows produce more PRL to keep equilibrium and maintain thermoregulation compared to NH cows (Collier et al., 2008). Stressed cows will experience increases in the production of PRL which in turn will increase the amounts of the receptors. Hence, it is important to understand PRLR expression in the skin of animals with different hair coat. Materials and Methods After shaving of the rump, skin biopsies (5mm) from dry Holstein cows (n=6 per treatment; SH and NH cows) were obtained and immediately stored in liquid nitrogen for subsequent RNA extraction. Skin samples were then homogenized using a Polytron and RNAzol (Molecular Research Center, Cincinnati, OH). Total RNA was isolated according to the manufacturer's instructions. RNA concentration and quality was measured using a nanospectrophotometer. Reverse transcription and Real Time PCR amplification were performed by using SYBR FAST kit KAPPA ONE-STEP qRT-PCR in a final volume of 20 ul with total RNA of 40ng. Reaction cycles consisted of the cDNA synthesis at 42 ° C for 5 minutes followed by inactivation of the RTMix at 95 ° C for 10 minutes. Further amplification was performed with 45 cycles of a denaturation step for 4 seconds at 95 ° C and an alignment and extension step at 60 ° C for 30 seconds. PRLR and Glycerolaldehyde-3-phosphate-dehydrogenase (GAPDH) were designed using PrimerQuest (Integrated DNA Technology, IDT). The PRLR primers were 5’GGAAGGAGCCAACATGAA-3’ and 5’-TCAGCGTGTAATTGGTAGG-3’ for forward and reverse, respectively. The GAPDH primers were 5’-CAGCGACACTCACTCTTCTAC-3’ and 5’-GGAAGTCAGGAGATTCTCAGT-3’ for forward and reverse, respectively. Results and Discussion The relative expression of PRLR was similar (P=0.17) between SH and NH cows. Numerically, the relative expression of PRLR in SH cows was 1.09 times greater than NH cows (Figure 1). The skin is one of the organs with the lowest expression of PRLR, and perhaps a greater number of animals were needed to detect statistical differences. Evidence has been found that supports 7 the existence of a major gene responsible for producing short, thin and slick hair; it is known to be a mutation in the chromosome 20, specifically in the PRLR (Olson et al., 2003). Since no differences in the expression of the gene itself were detected, maybe it is because it affects the function of the PRLR instead of its expression in the animal. Collier et al. (2008) compared production of PRLR during heat stress between SH and NH and concluded that SH produce more PRL than NH to maintain their thermoregulation. Another research studied PRLR mRNA expression and PRL concentration in the plasma between SH and NH which affected the hair cycle acting directly at cellular level where they used PRL inhibitors found out that as expression levels of total PRLR mRNA gradually decreased in skin, PRL concentration in plasma also decreased and fiber growth rates increased at the same time (Chunwang et al., 2010). In 2011, Alamer et al. determined that a direct relationship exists between PRL and the environment’s temperature; whereas the environmental temperature increased so did the concentration of PRL in the blood. A study where PRL was increased to induce follicle cycles during which PRLR expression changes were determined by ribonuclease assays suggested that the cycle-related patterns of PRLR expression were regulated by PRL, thereby modulating hormonal responsiveness of seasonally growing hair follicles (Nixon et al.,2002). Figure 1. Relative expression of prolactin receptor in Holstein cows with different hair coat Conclusion The expression of the prolactin receptor in the skin between slick hair cows and normal hair cows was similar (P<0.05). Further investigations should test if structural differences exist in 8 PRLR of SH and NH cows, which might increase the actions of prolactin in the skin of these animals. Cited Literature Alamer, M. (2011). The Role of Prolactin in Thermoregulation and Water Balance During Heat Stress in Domestic Ruminants. Asian Journal of Animal and Veterinary Advances, 6(12), 1153– 1169. Chunwang, Y., Lixin, D., Wei, Z., Xiaoping, K., & ZHIHAI, J. (2010). Expression of Prolactin Receptor mRNA after Melatonin Manipulated in Cashmere Goats Skin during Cashmere Growth. Asian- Australasian Jorurnal ofAnimal Science, 23(10), 1291–1298. Collier, R. J., Collier, J. L., Rhoads, R. P., & Baumgard, L. H. (2008). Invited review: genes involved in the bovine heat stress response. Journal of dairy science, 91(2), 445–454. Nixon, A. J., Ford, C. A., Wildermoth, J. E., Craven, A. J., Ashby, M. G., & Pearson, A. J. (2002). Regulation of prolactin receptor expression in ovine skin in relation to circulating prolactin and wool follicle growth status. The Journal of endocrinology, 172(3), 605–614. Olson, T. (2006). Evaluation and utilization of the Slick hair gene in Florida and Caribbean Dairies. USDA NIFA. Olson, T., Lucena, C., Chase, C. C., & Hammond, A. C. (2003). Evidence of a major gene influencing hair length and heat tolerance in cattle. Journal of Animal Science, 81(1), 80–90. 9 Use of exopolysaccharides producing lactic acid bacteria in the manufacture of low fat Gouda cheese Milton S. González and Leyda Ponce de León Department of Animal Science Abstract In different shapes, textures and flavors, dairy products has been the joy and the primary source of calcium, and other nutrients such as potassium and vitamin D in almost everyone’s daily diet. Among them all, cheese demand and consumption has increased all over America in the last decades. Particularly, Gouda cheese is one of the most popular cheeses in the world, by its dense texture and creamy flavor. Although, it is recommended to consume low-fat or fat-free dairy products, most of the regular products like Gouda cheese, contain many saturated fats and LDL (Low-Density Lipoprotein) cholesterols. If not eaten in the right portions can increase the risk for coronary heart diseases and diabetes. Also, the majority of low fat or fat free cheeses tend to be dryer, harder, and have a “rubbery” texture, which overshadows the possibility of becoming part of the consumer’s preference. Therefore, lactic acid bacteria are implemented as natural thickeners to improve the texture. The Streptococcus thermophilus ssp. strain is a high efficient thermophile organism that produces an exopolysaccharide secretion (EPS), an extracellular enzyme that works as a biological defense mechanism against environmental threats to the cell, especially against dehydration. The EPSs produced by S. thermophilus form a barrier that increase the viscosity and moisture, by preventing the cheese from draining much of the water that is lost in the molding and pressing process. Preliminary data shows that EPS cheeses have a greater compressibility in comparison with Non-EPS ones. Consequently, more desirable traits are found in the EPS low fat Gouda cheese, like a smoother, moister, and creamer texture than regular manufactured low fat Gouda cheese. Therefore, the EPS treatment is a potential alternative to improve the organoleptic qualities such as texture and mouthfeel in low fat Gouda cheese. Keywords: Low Fat Gouda Cheese, EPS, Texture Improvement Resumen En diferentes formas, texturas y sabores, los productos lácteos han sido el encanto y la fuente primaria de calcio, y otros nutrientes como potasio y vitamina D en la dieta diaria. Entre todos ellos, la demanda y el consumo de queso en Estados Unidos ha aumentado en las últimas décadas. Particularmente, el queso Gouda es uno de los quesos populares, por su textura densa y sabor cremoso. Aunque, se recomienda consumir productos lácteos reducidos en grasa o descremados, la mayoría de los productos regulares como el queso Gouda, contienen grasas saturadas y el colesterol LBD (Lipoproteína de Baja-Densidad). Si no se consumen en las porciones correctas puede aumentar el riesgo de enfermedades coronarias del corazón y diabetes. Además, la mayoría de los quesos bajos en grasa o descremados tienden a ser más secos, más duros, y tienen una textura "gomosa", que reduce la posibilidad de formar parte de la preferencia del consumidor. Por lo tanto, se implementan bacterias productoras de ácido láctico como espesantes naturales para mejorar la textura. La cepa de Streptococcus thermophilus ssp. es un organismo termófilo de alta eficiencia, el cual produce una secreción de exopolisacáridos (EPS), sustancia extracelular que funciona como un mecanismo de defensa biológico contra amenazas ambientales para la célula, especialmente contra la deshidratación. El EPS producido por S. 10 thermophilus forma una barrera que aumenta la viscosidad y humedad, previniendo así que el queso drene la mayor parte del agua que se pierde en el proceso de moldeado y prensado. Los datos preliminares muestran que quesos con EPS tienen una mayor capacidad de compresión en comparación con los quesos Sin-EPS. Consecuentemente, se encuentran más rasgos deseables en el queso Gouda bajo en grasa con EPS, como mayor humedad, y una textura más suave y cremosa que el queso Gouda bajo en grasa fabricado tradicionalmente. Palabras Claves: Queso Gouda Bajo en Grasa, EPS, Mejoramiento de Textura Introduction Lactic acid bacteria (LAB) have become an important component of dairy products industry, by its efficient fermentation qualities (Cogan et al., 2007). Two of the most common LAB used as starter cultures in cheese production are Lactobacillus delbrueckii spp. and Streptococcus thermophilus spp., both work effectively at a temperature range of 36-45°C. Despite their facultative differences, S. thermophilus spp. being aerobic, and L. delbrueckii spp. being anaerobic, they form a suitable combination for long term fermentation as they complement each other in the different stages of cheese manufacture. Moreover, S. thermophilus spp. is capable of synthesize exopolysaccharides (EPS), an extracellular polymers that may be assembled as capsular polysaccharide, or may form a slime layer loosely attached to the cell surface or secreted into the environment. (Madigan et al., 1997). EPS are theorized to protect bacteria against detrimental environmental conditions, also it has been shown to protect the cells against phage infection. (Kang and Cottrell, 1979). In addition, it has been thought that EPS produced by LAB have beneficial impact on human health such as cholesterol-lowering ability (Pigeon et al. 2002), immunomodulation, and antitumoral activities (Kitazawa et al., 1998; Chabot et al., 2001), and prebiotic effects. (DalBello et al., 2001; Korakli et al., 2002). One of the most important EPS attribute is its water-binding properties, which can be applied to reduced fat cheeses. It increases the moisture in the non-fat portion, interfere with protein-protein interactions and reduce the rigidity of the protein network, while increasing the viscosity of the serum phase (Hassan, 2008). Reduced-fat cheeses tend to be drier, and due to high casein content imparts a firm and rubbery body and texture (Emmons et al., 198; Mistry et al., 1993; Metzger and Mistry, 1995; Mistry, 2001). Thanks to the advances in biotechnology industry and food science, this EPS custom can be used as a substitute to the fat properties, providing consistency, moisture and lowering synergies (Ruas-Madiedo et al., 2005). This represents a reduction in the saturated fats and LDL (Low-Density Lipoprotein) content in cheese, while potentially lowering the risk to suffer coronary heart diseases, obesity and diabetes (USDA, 2011). Materials and Methods The project was conducted in a Milk and Dairy Products and Food Microbiology laboratories located in the Alfredo Ramírez de Arellano y Rosell building at the University of Puerto Rico, Mayagüez Campus. 11 Cheese Making Approximately three hundred and sixty liters of whole milk were obtained from UPRM Agricultural Experiment Station Dairy Farm in Lajas. The milk was divided into two portions of nearly one hundred and eighty liters and the fat percent was determined by Babcock method. After the fat percent determination to a 3.0%, one hundred and eighty liters are skimmed (figure 1), divided into two portions of ninety liters of skimmed milk, and standardize up to a 1.5% by calculating and adding 4.4 pounds of warmed skimmed cream to each portion by the Pearson Square method. First two 1.5% fat milk portions were pasteurized in two tanks at 64°C for thirty minutes, and let cool. At 42°C the Sacco® Lyofast commercial starter cultures were added respectfully to each tank: one with Non Exopolysaccharide-Producing Streptococcus thermophilus spp., and Lactobacillus delbrueckii spp. bulgaricus; and another with Exopolysaccharide-Producing Streptococcus thermophilus spp., Lactobacilus delbrueckii spp. bulgaricus, and Lactobacillus delbrueckii spp. lactis. Titulable Acidity tests were performed at this point to verify if the cultures were producing lactic acids. The animal rennet was applied to the milk, and let them set for about thirty minutes until it forms the clot. The cheese curds were cut with knives and cooked in the whey for thirty minutes at 42°C. Whey was drained from the tank and the cheese curds were directly salted. The cheese curds are placed in a rectangular mold with a cheese cloth, and were compressed using a hydraulic press for eighteen hours. The cheese blocks were vacuum packed and placed in a cava at 15°C for three months. These procedures were repeated with the 3.0% fat milk portions, excluding the skimming and standardizing procedures. Through ripening process, every fourteen days samples were collected and tested from each cheese block. Texture Analysis The texture firmness analysis consisted in cutting cheese in a wheel shape with about the same shape and sizes. These samples were kept at 25°C, were compress with a Pasta Adhesive Aluminum probe with measures of 50mm X 37mm with 1Kg of force, by a TX-XT2 Texture Analyzer that registered the peak positive distance that progress on the cheese sample surface, determining the hardness of the cheese. Moisture Analysis The moisture percent in cheese sample was determined by a Microwave Humidity/Solid Analyzer System by placing 2-3 grams of shredded cheese in fiberglass pads into the CEM machine. The cheese samples were dried and the differences in weight were determined. Microbial Enumeration In addition, a LAB Enumeration test was performed by homogenizing 11 grams of cheese sample in a 20% solution of sodium citrate. Serial dilutions were made using peptone water at 1%, and then were served in petri dishes using the pour plate technique. LAB were enumerated using MRS agar and incubation for 48 hours at 36°C in a anaerobic jar (Wehr, 2004). 12 Results and Discussion This study investigated the contributions of the EPS produced by Streptococcus thermophilus spp. and its properties to retain moisture and improve the texture of the low fat Gouda cheese. Analysis on 1.5% and 3.0% milk fat cheeses made with EPS-producers commercial starter cultures showed to increase the moisture levels through the ripening process, even though they lose some humidity through time. According to Figure 2, the EPS cheeses had the higher moisture values due to its water-binding property. Although, unexpectedly the 1.5% Milk Fat Non-EPS cheese had a sudden increment in its moisture level, perhaps this could result from a change in the environment humidity levels or an error from the CEM analyzer. Also a texture firmness analysis was performed by applying 1 Kg force over the cheese sample surface. As Figure 1 shows, 3.0% milk fat cheeses had a bigger peak positive distance through the first three weeks, and the they decrease to 4.296 mm and 2.279 mm by 56 days due to the available moisture and cream in cheese samples, while being EPS one the higher value. On the other hand, 1.5% milk fat cheeses in the first two weeks they remain constant, but from that point the NonEPS decreased its peak positive distance to a 0.706 mm by 56 days, while the EPS cheese increased its value to 1.716 mm, meaning a softer texture. Furthermore, LAB enumeration was made to ensure if the populations where in the specific range to carry the metabolic activities in the cheese. The LAB populations shown by table 1 presents bigger numbers of colonies for the 3.0% milk fat cheeses than 1.5% milk fat, while the EPS cheeses than Non-EPS ones. Thus to the higher levels of moisture and viscosity in the cheese from the EPS, a higher number of LAB may be found. Table 1. Lactic Acid Bacteria Enumeration in dilutionsof 10-7 Cheeses LAB Population 3.0% Milk Fat Non-EPS 1.28 x109 3.0% Milk Fat EPS 1.29 x109 1.5% Milk Fat Non-EPS 8.0 x108 1.5% Milk Fat EPS 8.39 x108 Figure 1. Gouda Cheese Texture Development 13 Figure 2. Humidity percentage in cheese samples Conclusions EPS-Producing LAB such as Streptococcus thermophilus spp. could be implemented to increase the moisture retention in low fat Gouda cheese, since also they were in the specific range of population to perform the metabolic activity. In Addition, the EPS helps to decrease the low fat Gouda cheese firmness. Thus EPS properties embodies positive traits for the dry and rubbery texture of the low fat Gouda cheese. 14 Cited Literature Chabot, S., H. L. Yu, L. De Léséleuc, D. Cloutier, M. R. van Calsteren, M. Lessard, D. Roy, M. Lacroix, and D. Oth. 2001. Exopolysaccharide from Lactobacillus rhamnosus RW-9595M stimulate TNF, IL-6 and IL- 12 in human and mouse cultured immunocompetent cells, and IFN-g in mouse splenocytes. Lait 81: 683-697. Cogan, T., Beresford, T., Steele, J., Broadbent, J., Shah, N., & Ustunol, Z. (2007). Invited Review: Advances in Starter Cultures and Cultured Foods. J. of Dairy Science, 90, 4005-4021. Dal Bello, F. D., J. Walter, C. Hertel, and W. P. Hammes. 2001. Invitro Studyof prebiotic properties of levan-type exopolysaccharides from lactobacilli and non-digestive carbohydrates using denaturing gradient gel electrophoresis. Syst. Appl. Microbiol. 91: 232-237. Emmons, D. B., M. Kalab, E. Larmond, and R. J. Lowrie. 1980. Milk gel structure X. Texture and microstructure in Cheddar cheese made from whole milk and from homogenized low fat milk. J. Texture Stud. 11: 15-34. Hassan, A. 2008. ASDA Foundation Scholar Award: Possibilities and Challenges of ExopolysaccharideProducing Lactic Cultures in Dairy Food. J. Dairy Sci. 91(4): 1282-1296. Kang, K. S., and I. W. Cottrell. 1979. Polysaccharide, p. 417-481. in H. J. Peppler and D. Perlman nd (ed.),Microbial technology: Microbial processes, 2 ed., Vol 1. Academic Press, Inc., New York. Kitazawa, H., T. Harata, J. Uemura, T. Saito, T. Kaneko, and T. Itoh. 1998. Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii spp. bulgaricus. Int. J. Food Microbiol. 40: 169-175. Korakli, M., M. G. Gänzle, and R. F. Vogel. 2002. Metabolism by bifidobacteria and lactic acid bacteria of polysaccharide from wheat and rye, and exopolysaccharides produced by Lactobacillus sanfranciscensis. J. Appl. Microbiol. 92:958-965. th Madigan, M. T., J. M. Martinko, and J. Parker. 1997. Brock Biology of Microorganisms. 8 ed. Prentice Hall International Ltd., London, UK. Metzger, L. E., and V. V. Mistry. 1995. A new approach using homogenization of cream in the manufacture of reduce fat Cheddar cheese. 2. Microstructure, fat globule distribution, and free oil. J. Dairy Sci. 78: 1883- 1895. Mistry, V. V. 2001. Los fat cheese technology. Int. Dairy J. 11: 413-422. Mistry, V. V., and D. L. Anderson. 1993. Composition in microstructure of commercial full-fat and lowfat cheeses. Food Struct. 12: 259-266. Pigeon, R. M., E. P. Cuesta, and S. E. Gilliland. 2002. Binding of free bile acids by cells of yogurt starter culture bacteria. J. Dairy Sci. 85: 2705-2710. Ruas-Madiedo, P., and De los Reyes-Gavilán, C. 2005. Invited Review: Methods for the Screening, Isolation and Characterization of Exopolysaccharide Produced by Lactic Acid Bacteria. J. Dairy Sci., 88(3), 843-843. USDA MyPlate Dairy Group -- All about the Dairy Group. (n.d.). Retrieved March 27, 2015, from http://www.choosemyplate.gov/food-groups/dairy.html Wehr, H., & Frank, J. (2004). Standard Methods for the Examination of Dairy Products (17th ed.). Washington DC: American Public Health Association. 15 The effect of scrotal temperature on semen quality parameters in Holstein Friesian and Jersey bulls Camille Cordero-Aponte and Jaime E. Curbelo-Rodríguez Department of Animal Science Abstract In Puerto Rico, the use of exotic bovine dairy breeds to increase milk production has been commonly implemented for decades. High temperatures and humidity, characteristic of our island, have been reported to adversely affect the reproductive performance of bulls. This study was conducted to evaluate the effect of scrotal temperature on semen quality parameters in Jersey and Holstein-Friesian bulls. Jersey and Holstein bulls (n=3/breed) were evaluated. Scrotal surface temperature was determined in different anatomical locations of the scrotum [i.e., proximal (P), medial (M) and distal (D)] using infrared thermography. Several semen quality parameters were evaluated in each bull using the computer-assisted sperm analysis system (HT CASA II). No significant differences between semen quality parameters among bull breeds where observed except for proximal (p=0.0153) and distal droplet (p=0.0405), which were superior in Jersey bulls. Scrotal temperatures of Jersey bulls were significantly lower than Hosltein bulls with Mmin of 32.7± 0.07°C vs 34.35 ± 0.30 °C (p=0.005) and Davg 32. 72 ± 0.47 vs 34.51± 0.42 °C (p= 0.0547), respectively. The results found in this study suggest that Jersey bulls posses superior reproductive capacity than Holstein bulls, in part by their superior scrotal thermoregulatory capacity. Keywords: Semen quality, scrotal surface temperature, thermography imaging Resumen En Puerto Rico, el uso de razas lecheras bovinas exóticas, empleadas para incrementar la producción de leche, ha sido implementado durante décadas. Altas temperaturas y humedad, características de nuestra isla, tienen impacto adverso sobre la capacidad reproductiva de los toros. Este estudio se realizó para evaluar el efecto de la temperatura escrotal sobre los parámetros de calidad del semen en toros lecheros. Se evaluaron toros de la raza Jersey y Holstein (n = 3 / raza). Se determinó la temperatura escrotal en distintas localidades anatómicas del escroto [i.e., proximal (P), medial (M) y distal (D)] utilizando termografía infrarroja. Varios parámetros de calidad de semen se evaluaron en cada toro utilizando el sistema de análisis de semen (HT CASA II). En resumen, no se observaron diferencias significativas en parámetros de calidad del semen entre razas de toros, a excepción de la gota citoplásmica proximal (p = 0.0153) y la gota citoplásmica distal (p = 0.0405), las cuales fueron superiores en los toros Jersey. Temperaturas escrotales de toros Jersey fueron significativamente menores que la de toros Holstein, con un Mmin de 32.7 ± 0.07 °C vs 34.35 ± 0.30°C (p = 0.005) y Davg 32. 72 ± 0.47 vs 34.51 ± 0.42 °C (p = 0.0547), respectivamente. Los resultados encontrados en este estudio sugieren que los toros Jersey poseen capacidad reproductiva superior a toros Holstein, en parte por su mayor capacidad termoregulatoria escrotal. Palabras Claves: Calidad de Semem, temperatura de superficie escrtola, termografia 16 Introduction The geographical position of Puerto Rico is described as tropical climate governed by high temperatures and humidity throughout the year. The dairy industry of our island is the most important economically. Dairy farmers have implemented different genetic strategies such as the use of exotic dairy breeds from temperate regions (e.g., Europe) to increase milk production. It has been reported that high ambient temperatures have a negative effect on the productive performance of cattle, whether a limitation on milk production, pregnancy rate, growth rate, and in the case of dairy bulls, their reproductive capacity and semen quality. Research studies have found a relationship between heat stress and semen quality parameters (Rios et al. 2013). In Puerto Rico there is insufficient or no information on semen quality parameters and reproductive capacity of local dairy bulls. This information is of relevant importance to increase reproductive efficiency in local dairy farms Lunstra and Coulter (1993) found that infrared thermography of scrotal surface is a useful tool for determining the reproductive potential of diary bulls and that there was a clear association between the estimation of scrotal thermoregulation and the quality of semen and fertility in bulls. Similarly, it has been found that higher temperatures cause testicular degeneration (Rios et al. 2013). That is why, it is expected that high temperatures in which bulls are constantly exposed will cause a negative effect on semen quality parameters and therefore a decrease in their reproductive capacity. Therefore, the aim of this study was to evaluate the effect of scrotal temperature on semen quality parameters in Jersey and Holstein Friesian bulls. Materials and Methods Two dairy bull breed from two commercial dairy farms were used. The dairy "Tai South Farm" provided 3 Jersey bulls and the dairy "Enriqueta" provided 3 Holstein Friesian bulls. Both dairies are located in Lajas, Puerto Rico. Each bull was restricted individually in chute (Fig. 1 panel A). Once restrained, infrared thermography of the scrotal surface was performed on each bull (Fig. 1 panel B). The thermal camera (T8 FLIR) was positioned 3ft from the rear of each bull. Also, certain observations were made and in case of any presence of blood or wounds annotations were made. Then, using an electroejaculator, a semen sample was obtained from each bull (Fig. 1 panel C). Once each sample was collected, an extender formula, previously prepared in the Reproduction Laboratory, was applied in a 1:1 ratio, and time of collection and application of extender was annotated. Then, a questionnaire was provided to complete information including animal management practices and animal characteristics (data not shown). The samples were then transferred to the Molecular Genetics and Musculoskeletal Biology laboratory at the Department of Animal Science. Each sample was diluted using a micropipette in a 1:10 ratio with a buffer solution. Three microliters from the sample dilution was obtained 17 and placed in a Leja 4 Chamber 20 Micron Slide. Finally an analysis of semen quality parameters was performed using the HT CASA II system. Figure 1. Restrainment of bulls (panel A), infrarred thermography imaging of scrotal surface (Panel B), and semen collection (Panel C) of bulls included in the study. Results and Discussion Sperm motility parameters The Static parameter represents the non-moving sperm cells in a semen sample. No significant differences (p0.4365) were observed between breeds (Fig. 4). A possible explanation for this is the high variability observed (SE ± 25.0549 and 8.6845) for Holstein and Jersey bulls respectively. The Progressive parameter considers sperm moving with straightness and an average path velocity. No significant differences were detected (p=0.6939). This could also be explained by the variability observed between Holstein bulls (SE ±7.5810) and Jersey bulls (SE ± 4.3552) (Fig. 4). The Motile parameter denotes a sperm that moves more than its head length from its original position due to acquisition. In this case as well, there were no significant differences (p=0.4365). High variability observed in Holstein and Jersey bulls (SE± 25.0549 and 8.6845, respectively) could be a possible cause for no observable significant differences (Fig. 4). Lastly, the slow motility parameter represents a sperm moving with less than the minimum required for a curvilinear velocity or average path velocity. No significant differences were observed (p=0.6596) among breeds. Sperm morphology parameters Bent tail represents any sperm with an exceeding bending rate. No significant differences were observed (p=0.8176) between Holstein and Jersey bulls. Sperm are categorized as having Coiled Tail if the tail bends 180º or more over its length. No significant differences were observed (p=0.6432) between breeds. The DMR (Distal Midpiece Reflex) indicates if the tail of the sperm is wrapped around a distal cytoplasmic droplet. In this study, no significant differences were observed (p=0.5994). The Distal Droplet indicates the presence of a cytoplasmic droplet further down the tail from the base of the head while the Proximal Droplet represents a cytoplasmic droplet that is attached to the midpiece or base of the head of the sperm. In both cases, these are considered as defects and immaturity in sperm. Significant differences were observed (p= 0.0405 and 0.0153) 18 in the Distal and proximal droplet parameters respectively, between both breeds. These significant differences might be associated with abnormal scrotal temperatures exhibited in bulls. Normal Sperm fraction and sperm concentration The Normal fraction values show significant differences between both breeds (p= 0.0153). This value might be associated to the differences observed in scrotal temperatures (lower in Jersey). The Motile concentration is derived from motile cells only and the Total concentration includes the concentration of all parameters. In this study no significant differences were observed in both cases with motile concentration having p= 0.1051 and total concentration having p= 0.1961. In the case of total concentration a high variability can be observed between Jersey (SE± 343.6) and Holstein (SE± 16.0307) bulls (Fig. 6). Table 1- Semen quality parameters percentages of motility for Jersey and Holstein bulls individually and their average percentages Breed Parameters Percentage (%) Jersey 1 Static 28.0 Progressive 11.8 Motile 72.0 Slow 11.5 Jersey 2 2.2 26.7 97.8 2.2 Jersey 3 Average 1.7 10.63 21.3 19.93 98.3 89.36 2.8 5.5 Holstein 1 Holstein 2 10.3 6.7 20.5 26.7 89.7 93.3 2.6 6.7 Holstein 3 83.6 1.5 16.4 13.4 Average 35.53 16.23 66.46 7.56 Table-2 Semen quality parameters percentages of morphology for Jersey and Holstein bulls individually and their average percentages Breed Jersey 1 Jersey 2 Jersey 3 Average Holstein 1 Holstein 2 Holstein 3 Average Parameters Percentage (%) Bent Tail Coiled Tail DMR Distal Droplet Proximal Droplet 13.5 0.0 2.6 5.36 1.3 6.7 11.9 6.63 5.1 0.0 0.3 1.8 2.6 0.0 0.0 0.86 0.7 2.2 0.0 0.96 0.0 0.0 1.5 0.5 4.5 6.7 6.4 5.86 3.7 0.0 2.4 2.03 9.1 2.2 15.3 8.86 29.5 46.7 31.3 35.83 19 Infrared thermography imaging of scrotal surfaces Scrotal temperature was measured on three different anatomical locations: proximal, medial and distal. Three different values, the maximum, minimum and average temperature were obtained for each section of the scrotal surface of the bulls. For the proximal variables, the Pmax (p=0.4847), Pmin (p=0.3642 ) and Pavg (p=0.3642 ) show no significant differences between the breeds evaluated. Medial temperatures shown no significant differences were observed for the Mmax (p= 0.6184). Significant differences were observed for the Mmin (p=0.055), but no significant differences were observed for Mavg (p=0.1739). For distal variables, the Dmax (p=0.1734) and the Dmin (p=0.1245) demonstrated no signficiant differences between breeds, but in the case of Davg (p=0.0547) significant diffrences were observed. These values in whicha significant difference can be noticed suggest that Jersey bulls have thermoregulatory advantage over Holstein bulls. Percentages (%) 120 100 80 60 40 Jersey 20 Holstein 0 Semen Quality Parameters of Motility Figure 2: Sperm motility parameters between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. Percentages(%) 45 40 35 30 25 20 15 10 5 0 * * * Jersey Holstein Semen Quality Parameters of Morphology Figure 3: Sperm morphology parameters between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. 20 1400 Concedntrartion M/ml 1200 1000 800 Jersey 600 Holstein 400 200 0 Concentration Figure 4: Sperm concentration parameters between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. Maximum Infrared Temperature (C±°) 38 37 36 35 34 33 32 31 30 Jersey Holstein Proximal Medial Distal Anatomic Scrotal Locality Figure 5: Anatomic scrotal locality maximum temperature readings between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. Minimun Infrared Temperature (Cº) 37 36 35 34 33 32 31 30 29 28 Jersey Holstein Proximal Medial Distal Anatomic Scrotal Locality Figure 6: Anatomic scrotal locality minimum temperature readings between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. 21 * Average Infrared Temperature (C°) 38 37 36 35 34 * 33 32 Holstein 31 30 29 Jersey Proximal Medial Distal Anatomic Scrotal Locality Figure 7: Anatomic scrotal locality average temperature readings between Holstein (purple) and Jersey (gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in * p=values <0.05. Conclusion In this preliminary study, there was an evidently high varibility in sperm quality paramters between Holstein-Friesian and Jersey bulls. The results confirm an association between scrotal temperatures in bulls and semen quality parameters. Higher scrotal temperatures in bulls suggest a larger parcentage of abnormalities in sperm such as, bent tail, coiled tail, DMR, and proximal and distal droplets. Specifically there were significant differences in distal and proximal droplet parameters between these two breeds. In addition, Jersey bulls show numerically larger sperm concentration than Holstein bulls. Despite the fact that both breeds’ reproductive capacity could be adversely affected by high scrotal temperatures, the differences in abnormalities could imply that Jersey bulls might be a more suitable breed for reproduction in tropical and subtropicl regions in comparison to the Holstein Friesian breed. Cited Literature Fiaz, M., Usmani, R. H., Abdullah, M., Ahmad, T. 2009. Evaluation of semen quality ofHolstein Friesian and Jersey bulls maintained under subtropical environment. PakVet J, 30(2):75-78 Lunstra, D. D., Coulter, G. H., 1993. Scrotal Thermography as a tool for predicting semen quality and natural-mating fertility in young beef bulls. Ríos, V. C., Ortiz, N. M., Valencia, A. F., Orjuela, J. A., 2013. Estrés calórico y ysu relación con variables reproductivas en machos bovinos en la Amazonia Colombiana. Revista electrónica de Veterinaria. Retrieved from http://www.veterinaria.org/revistas/redvet/n040413/041309.pdf 22 The retained placenta malady in a Puerto Rican dairy cattle herd: prevalence, treatment and effects on animal performance Nicole M. Irizarry, Paul F. Randel, Héctor Santana and Ricardo Montañez Department of Animal Science Abstract Retained placenta (RP) is a reproductive dysfunction universally present in dairy cattle worldwide. In Puerto Rico it has received rather little research attention. The present work, conducted in the dairy herd of the University of Puerto Rico at Mayaguez, involves one phase of experimentation, in which two medical procedures for treating RP are being compared: (T1) hormones (oxytocin and prostaglandin) to expedite expulsion of the placenta plus antibiotic and (T2) hormones only; with the object of determining if the antibiotic is really necessary to treat routine cases of RP or if its use is not cost effective. Of the 68 parturitions that took place in the herd from the start of experimentation, in Sept. 2014, until the present writing, in mid Jan. 2015, only 6 cows retained the placenta for 24 hours or more (8.82% of incidence) of which 3 received T1 and 3 received T2. Data collection will continue throughout the current academic semester. The second phase of the work will involve a search of the herd records, covering the years 2012 to 2014 and part of 2015, to determine the overall prevalence of RP and possible differences in prevalence due to the independent variables: parturition number (first vs. second or later), season of the year (hotter vs. cooler months), and perhaps breed group (pure or high grade Holstein vs. crossbred). Also of interest, is the effect of RP on subsequent reproductive performance as judged by criteria such as interval until the next parturition, interval from calving to the next service and number of inseminations to achieve pregnancy. Keywords: Retained placenta, Dairy cattle, Antibiotic treatment Resumen La retención de placenta (RP) es un problema reproductivo comúnmente presente en el ganado lechero a nivel mundial. En Puerto Rico este tema ha recibido muy poca atención en el ámbito científico. Este trabajo, llevado a cabo en el hato lechero de la Universidad de Puerto Rico en Mayagüez, envuelve una fase experimental, en donde dos tratamientos veterinarios para tratar la RP están siendo comparados: hormonas oxitocina y prostaglandina para acelerar la expulsión de la placenta con adición de antibiótico (T1) y hormonas exclusivamente (T2); con el objetivo de determinar si el antibiótico es realmente necesario para tratar casos rutinarios de RP o si su uso es o no costo efectivo. De los 68 partos ocurridos desde el inicio del experimento en septiembre 2014 hasta el presente en el mes de enero 2015, 6 vacas retuvieron la placenta por 24 hrs o más (8.82% de incidencia) de las cuales 3 recibieron T1 y 3 recibieron T2. La recolección de datos continuará por lo que resta de este semestre académico. La segunda fase del experimento envuelve una búsqueda en los récords de la vaquería desde el 2012 hasta el 2014 y parte del 2015 para determinar la incidencia total de RP y diferencias en estos casos debido a variables independientes, tales como: número de partos (primer parto vs. partos múltiples), época del año (meses calientes vs. meses frescos), y la raza (Holstein puras vs. cruce de razas). También se desea conocer el efecto de RP en el futuro desempeño reproductivo de la vaca evaluando el intervalo entre partos, intervalo entre un parto y el siguiente servicio, y el número de inseminaciones requeridas para lograr la preñez. Palabras Claves: Retención de placenta, Ganado lechero, Antibióticos 23 Introduction After calving, the cow usually expels the placenta in the first 24 hours by the action of hormones naturally released in the organism to degrade the placenta and stimulate uterine contractions. If the placenta is not expelled properly, leaving remanents inside the system for a long time, an infection could develope. The retained placenta makes the perfect medium for bacteria to grow, causing the bacteria present in the organism combined with the bacteria that enter the system at the moment of calving to develop a massive infection. These infections are designated as metritis or endometritis at varying levels of severity, which are different types of uterine infections. Some signs of infection are: strong stench, uterine discharges with a brownish red color, poor appetite, decrease production, absence of striations due to the low rhythm of uterine contractions and unstable temperature or fever in severe cases. Chronic or continuous cases of metritis affect the fertility of the cow; in detriment to the production and eventually, the farm income. One of the causes associated with the RP problem is the diet during the dry period. The nutritional factor during this period is vital, providing the cow with the necessary nutriments for the development of the embryo. In the south of Puerto Rico, pastures are usually of low quality in certain periods of the year, especially in the dry seasons, which may be a causal factor. Different methods exist to treat animals with retained placenta: uterine lavage, antibiotic or hormone administration, or both; the administration of hormones being the normal protocol followed in cases of RP in the studied herd. The usage of antibiotics is limited because they are expensive and bacteria can turn resistant to them if its usage is constant. Based on this fact, it was expected that the addition of the antibiotic in this research would result in no marked impact on the recovery and prognosis of a cow with retained placenta, which justifies reducing the usage of antibiotics in routine cases of RP. Materials and Methods Antibiotic treatment versus Hormones only treatment The study was conducted in the dairy operation at the Agricultural Research Station of the University of Puerto Rico, Mayaguez Campus, at Lajas, Puerto Rico. Cows and heifers about to calf were kept under regular management for pregnant dairy females. After calving, if the animal expelled the placenta in the first 24 hours it joined the reference group with no treatment. If in 24 hours the cow had not expelled the placenta, it was assigned to the antibiotic treatment or the control group (hormones only). Data were collected during weekly visits (Figure. 2) and the attending veterinarians did the post-partum examinations every two weeks. Individuals under the first treatment (T1) received an intramuscular injection twice daily for a 5day period of 5.00 ml of oxytocin and 5.00 ml of prostaglandin F2α with an addition of a subcutaneous injection of antibiotic (Excede). Individuals under the second treatment (T2) or control group received only oxytocin and prostaglandin in the same dosages. The parturitions considered for inclusion in the research were all that occurred in the months of September through January. 24 Figure 1. Expelled Placenta This image shows a placenta on the ground expelled normally. Figure 2. Field Data Collection Body and behavior observations were taken as part of the weekly data collection 25 Incidence from 2012 to 2014 Data were collected from the PC-Dart and Madero Dairy System (AFI Milk) programs, in the digital records of the farm for the 2012 to 2014 years interval. Results and Discussion Treatment with antibiotic vs. control treatment Data from the cows that retained placenta during the experiment and were assigned either to T1 or T2. Of the 68 parturitions at the moment of summarizing, 8.82% of the cows retained the placenta. Table 1. Cows under T1 and T2. Cow ID T1 or T2 Calving Date (m/d/yr) Post-calving Live Weight (lb) Calf Survival Days Retaining Placenta Post-partum Examination 209 T2 09/10/14 879 L 2 273 T1 09/15/14 1.039 L 9 302 T2 10/08/14 - D 11 Mastitis possibly associated with placental retention Retained remanents of the placenta discovered several days later Metritis and mastitis 61 T2 12/06/14 1,215 L 6 134 T1 12/07/14 1,084 D 5 298 T1 1/20/15 950 D 7 Acute metritis; 50 days of infection. No complications after freemartin and twin male birth Post-partum weakness following dystocia Figure 3. Cow Weakened by metritis. The backbone of this very lean animal can be appreciated easily. The severe infection reduced her appetite and production 26 Figure 4. Infected cow. This is a dystocic cow straining to expel the placenta, that several days later, developed a chronic infection. Figure 5. Uterine discharge. Cow that calved normally with uterine discharge several hours later. 2012-2014 Incidence Table 2. Percentage of retained placenta cases from 2012-2014 Year # of Calvings # of RP cases RP % 2012 26 7 26.92 2013 2014 84 127 11 21 13.10 16.54 Total 237 39 16.46 Most of the cases from 2012 were treated with antibiotics. In 2013, the standardized protocols or RP cases at this farm changed to the usage of hormones, which it is still the norm. No dairy herd is completely free of RP problems, however good reproductive management and optimal feeding help to minimize this malady. “Metritis affects as many as 25% of the roughly 9 million dairy cows in the United States, costing nearly $400 per case in lost productivity and 27 treatment costs.” (Cornell University, 2011). The overall incidence found in the present research from 2012 to January 2015 was 16.33%. Comparing our results with the overall mean for the United States, the incidence found is proportionally higher for a small herd. A similar experiment was conducted in the State of Aguascalientes, Mexico (Wonchee, Z. et.al., 2002) based on the observation of 668 parturitions, from which 243 had RP and were treated with different combinations of prostaglandin, gonadotropins and oxytetracycline. Although the present data are too limited for firm conclusions, the Wonchee research concluded that antibiotic treatment has no significant advantage in RP cases; furthermore these authors also found no advantage from use of the hormones either. Conclusion Further data are needed to confirm conclusions of this study, but tentatively it appears that antibiotic use in routine cases (with no other complications) of retained placenta can be eliminated with a saving in costs. The incidence of retained placenta in the research herd has slightly increased in the past two years, as the herd size has doubled, being 16.46% for the overall 2012-2014 period. This means that further attention and improved management is needed to reduce the incidence of retained placenta to as low a level as possible. Cited Literature Cornell researchers create first metritis vaccine to protect dairy cows. 25/10/2014, of Cornell University, College of Veterinary Medicine. Web: http://www.vet.cornell.edu/news/metritis.cfm Wonchee, Z., Lozano, R., González, E., (2002). Evaluation of different treatments used in the early postpartum in dairy cows with metritis. Téc Pecu Méx, 40 (1), 105-117. 28 Soil nitrogen mineralization in soils cropped to maize (Zea Mays) of the southern semiarid zone of Puerto Rico Abdiel Santana, Johannies Rivera-Zayas and David Sotomayor-Ramírez Department of Crops and Agro-environmental Sciences Abstract Quantified soil nitrogen (N) mineralization rates under controlled conditions can be used to estimate rate constants and potentially mineralizable N pools. This information can be used to guide fertilizer-N management in crops and improve agroecosystem sustainability. Soils from the Jacaguas series (Fluventic Haplustolls) and Guamaní series (Torrifluventic Haplusteps) were sampled from plots of experimental fields evaluating the effects of fertilizer-N on maize yield. The Jacaguas soil was under a three-year maize maize-cowpea (Vigna unguiculata) rotation and Guamaní was under a fallow-maize cropping system. Soils were sampled from plots historically fertilized with 120 kg N/ha under fallow-maize and additional samples were gathered from Jacaguas soil under a cowpea rotation. Soils were air-dried and sieved to pass a 2-mm sieve and packed to selected bulk density in mesocosms. Fresh cowpea residue at a rate of (125 kg N/ha) was added to soils. The Guamaní and Jacaguas soils were incubated with (AM) and without (FA) cowpea amendment and the Jacaguas soil under the cowpea rotation was incubated without cowpea amendment to evaluate the residual soil N mineralization (CC). A 20-week laboratory incubation leaching experiment was performed. Controlled leachings were made at 0, 1, 2, 3, 4, 6, 8, 12, 16 and 20 weeks, and analyzed for inorganic N (NH4+-N and NO3-N). We report on results of data gathered up to 6 weeks. Soils amended with cowpea (AM) or with a history of cowpea rotation (CC) had a higher N accumulation of inorganic N (Nmin) at 6 weeks than unamended soils. The Jacaguas soil with A or CC treatments had higher rates of N mineralization during the early stages (0 to 3 weeks) but lower cumulative Nmin at 6 weeks than Guamaní soil. Cumulative Nmin at 6 weeks was in the order of Guamaní (AM) > Guamaní (FA) > Jacaguas (AM) = Jacaguas CC > Jacaguas (FA), with values of 58, 48.6, 39.2, 35.2, 25.3, mg N/kg, respectively. The results gathered to date suggest that amending soils with a covercrop residue such as cowpea is an efficient way to provide soil N to a crop and optimize the use of N fertilizers. Keywords: Soil Nitrogen, Nitrogen Mineralization, Nitrogen Management Resumen La cuantificación de las tasas de mineralización de nitrógeno (N) en el suelo bajo condiciones controladas puede ser usada para estimar las constantes de las tasas de mineralización y las reservas de N potencialmente mineralizables. Esta información puede ser usada como guía para el manejo de fertilizante nitrogenado en cultivos and y mejorar la sustentabilidad agro-ecológica. Suelos de la serie Jacaguas (Fluventic Haplustolls) y serie Guamaní (Torrifluventic Haplusteps) fueron muestreados en parcelas de campos experimentales donde se evalua los efectos de fertilizante N en rendimientos de maíz. El suelo Jacaguas estaba bajo un sistema de rotación maíz maíz-caupi (Vigna unguiculata) por tres años y el suelo Guamaní bajo un sistema de siembra-barbecho con maíz. Las muestras de suelos fueron tomadas de parcelas con historial de fertilización de 120kg/ha bajo sistema maíz-barbecho, además se tomaron muestras adicionales del suelo Jacaguas bajo sistema de rotación con cobertor caupi. Las muestras tomadas fueron 29 secadas al aire y luego cernidas con un tamiz de 2mm para ser empacadas a una densidad aparente seleccionada en mesocosmos. Material fresco de caupi fue agregado a los suelos a una tasa (125 kg N/ha). Los suelos Guamaní y Jacaguas fueron incubados con una enmienda de caupi (AM) y sin enmienda (FA) y el suelo Jacaguas bajo historial de rotación con caupi (CC) fue incubado sin enmienda para de este modo evaluar la mineralización del N residual. Se llevó a cabo un procedimiento de incubación y extracción de 20 semanas en el laboratorio. Extracciones controladas fueron llevadas a cabo en 0, 1, 2, 3, 4, 6, 8, 12, 16 y 20 semanas, y estas fueron analizadas para N inorgánico (NH4+-N y NO3-N). Se reportan resultados de data colectada hasta la sexta semana. Suelos enmendados con caupi (AM) o con historial de rotación de caupi (CC) tuvieron mayor acumulación de N inorgánico (Nmin) a las 6 semanas, en comparación a los suelos sin enmendar. El suelo Jacaguas con tratamiento A o CC tuvo mayores tasas de mineralización durante etapas tempranas (0 a 3 semanas) pero menor Nmin acumulado a las 6 semanas que el suelo Guamaní. Nmin acumulado a las 6 semanas fue en orden Guamaní (AM) > Guamaní (FA) > Jacaguas (AM) = Jacaguas CC > Jacaguas (FA), con valores de 58, 48.6, 39.2, 35.2, 25.3, mg N/kg, respectivamente. La data colectada hasta la fecha sugiere que enmendar suelos con residuos de un cobertor como caupi es una manera eficiente de proveer N del suelo al cultivo y optimizar el uso de fertilizante nitrogenado. Palabras Claves: Nitrógeno del Suelo, Mineralización de Nitrógeno, Manejo de Nitrógeno Introduction Soil N mineralization is a process in which organic matter is degraded into its mineral constituents, therefore making soil inorganic N available to plants. Mineralization occurs continuously under natural soil conditions (temperature, humidity, pH, aeration), but the two most influencing factors are temperature and soil water content (Cabrera, 2007). The quantification of soil N mineralization rates under controlled conditions can be used to estimate rate constants and potentially mineralizable N pools. Quantified mineralization rates can help understand how and when N is becoming available, since N becomes available gradually from organic matter we can measure how it becomes available from mineralization processes throughout time. By quantifying cumulative soil N we can also estimate mineralizable N pools, taking in consideration factors like the soils organic matter content, temperature, water content and other less influential but important characteristics. This information can be used to guide fertilizer-N management in crops, by determining the soils capacity to provide inorganic N and minimizing the amount of N fertilizer applied. By identifying the moment in which soil N is available and the moment of critical nutrient need in the plant, it is possible to synchronize the stated and have a better use of the N pool available from the soil to the crop. Determination of N available from the soil can also help better understand the N Cycle. This knowledge can lead to better N management to avoid contamination by excessive application of fertilizer-N, while also enhancing the soils microbiology, therefore improving agro-ecosystem sustainability. The latest years various multinational biotechnology companies dedicated to production of seeds have established in the southern semi-arid zone of Puerto Rico. This area is one of the major agricultural lands in the country. This zone has highly fertile land and it is cropped to a variety of 30 crops including but not limited to maize. An optimum management of this lands soil can result in better crop production and a more sustainable and eco-friendly agriculture. A 20-week soil incubation leaching experiment was performed, under controlled laboratory conditions. Soils from the Jacaguas series (Fluventic Haplustolls) and Guamaní series (Torrifluventic Haplusteps) were sampled from plots of experimental fields evaluating the effects of fertilizer-N on maize yield on DOW Agroscience facilities at Guayama and Santa Isabel, PR. The Jacaguas soil was under a three-year maize maize-cowpea (Vigna unguiculata) rotation and Guamaní was under a fallow-maize cropping system. Soils were sampled on both soils from plots historically fertilized with 120 kg N/ha under fallow-maize and additional samples were gathered from Jacaguas soil under a cowpea rotation. Soils were prepared by first being air-dried and then sieved to pass a 2-mm sieve. Samples were then packed to selected bulk density in mesocosms. Fresh cowpea residue at a rate of (125 kg N/ha) was added to both soils. The Guamaní and Jacaguas soils were incubated with (AM) and without (FA) cowpea amendment and the Jacaguas soil under the cowpea rotation was incubated without cowpea amendment to evaluate the residual soil N mineralization (CC). Objectives Quantify soil N mineralization to estimate rate constants and potentially mineralizable N pools. Understand cover crop rotation and cover crop amendment role in soil N mineralization processes. Materials and Methods Sampling depth was from 0 to 20 cm over each sub-plot and taken to the University of Puerto Rico at Mayagüez. Samples were air-dried and sieved through a 6mm mesh to then be mixed and homogenized. Soil pH with a 2:1 water ratio, soil moisture by the gravimetric method and soil texture (Kettler, 2001) were determined for each soil. Samples were packed using 60 ml syringes as incubation vessels. Syringes were weighted and a filter composed of 0.4g of celite and a 3.0cm Whatman® micro-fiber filter was added before weighting again. An amount of 40g of the prepared soils were added to each syringe, by treatment, and packed to selected bulk density in mesocosms, finally then total weigh was taken. Table 1. Experimental Treatments Location Soil management Guayama Fallow-maize Santa Isabel Treatment Descriptor None FA Fallow-maize Cowpea amendment AM Fallow-maize None FA Fallow-maize Cowpea amendment AM Cowpea-maize None FA Cowpea (+ Cowpea) amendment equivalent to 125kg/ha Control sample to measure N provided by the environment or any other external agent 31 Controlled leachings were made at 0, 1, 2, 3, 4, 6, 8, 12, 16 and 20 weeks based on Stanford & Smith (1972) and Cabrera & Kissel (1988) with modifications. Inorganic N extractions were made using 80mL CaCl2 0.01M, leaching for 3 hours on the extraction system (Zero-Max® Drive Power Block). A digital scale was used to weight samples after each step of the process. Leached solutions were poured through funnels with filter paper 25mL of each solution sample were poured into 25mL bottles. For each sample 2 drops of concentrated sulfuric acid were added and then they were refrigerated until analyzed and for inorganic N (NH4+-N and NO3-N. A 10mL of N free solution (340 mg CaSO4, 120 mg MgSO4, 15.04 mg K2SO4, 2.19 mg KH2PO4, 1L H2O) were added using the leaching system for 30 minutes to replace exchangeable ions in the soil sample. The samples were placed in a suction system for 4 hours applying suction at 0.33 kPa to get and appropriate water filled pore space (WFPS), leaving the soils around field capacity. Soil samples were then incubated for the next leaching. Optimum mineralization temperature was monitored and kept at 35°C (Kirschbaum, 1994) and water content was manipulated and kept around field capacity (Paul et al., 2000). Results and Discussion Results up to six weeks are reported. Pre-plant soil fertility parameters (Table 2 & 3) show both soils have comparable inorganic nutrient availability as organic matter (OM) content. OM, around 2%, indicates availability of mineralizable soil N. It is evident when comparing these parameters (P ppm & K ppm) to initial extraction values (T0) that the Jacaguas (SI) soil has had a longer N fertilizer application than the Guamani (G) soil. Table 2. General soil fertility pre-plant parameters of Guayama pH OM Bray 1 Ca Mg % 7.65 K Na CEC 48 25.5 -----P ppm---- 2.7 9 3521 867 166 Table 3. General soil fertility pre-plant parameters of Santa Isabel Treatment pH Bray 1 Olsen -----P ppm---71.43 36.18 Ca 7.65 OM % 2.08 3445 Mg K Na CEC ----------------------meq/100g-------------620 260.5 89.75 23.43 Fallow Cover Crop 7.65 1.88 49.68 3873 613.25 29.09 192.25 78 25.33 Extraction values for T0 reveal a substantial difference between Nmin at both locations, for the G soil values are around 6 mg N/kg soil while at the SI soil which has a longer history of N fertilizer values start at 48.7 and reach up to 140.6 mg N/kg soil. (Compare with Chart 1 & 2). Nmin results on chart 2 were expected, values are in the order of SI- CC, SI- AM, and SI-FA. This shows that CC rotation applied to this soil in combination with fertilizer N have led to the 32 highest amounts of accumulated Nmin, its was followed by AM which had higher values than FA due to N afforded by the amendment, but lower than CC due to N not being immediately available from the OM applied. This is supported by the approximate 4 year history of N fertilizing. Chart 1. Initial Extraction Guayama T 0 GUAYAMA Inorganic N (mg/kg soil) 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 6.6 6.2 0.0 Guayama FA Guayama AM Chart 2. Initial Extraction Santa Isabel T 0 SANTA ISABEL Inorganic N (mg/kg soil) 160.0 140.6 140.0 120.0 100.0 72.5 80.0 60.0 48.7 40.0 20.0 0.0 Santa Isabel FA Santa Isabel AM Santa Isabel CC 33 The Jacaguas soil had higher rates of N mineralization during the first 3 weeks when compared to the Guamani soil. Rates of Mineralization on the G soil started rising at the third week, surpassing the SI soil, but have not yet stabilized at six weeks. SI rates seems to be reaching Vmax values, but further data needs to be gathered form the following weeks to confirm this. N Mineralization rates are expected to reach Vmax by the 8th week of incubation. As observed in chart 2.1, Km values for the SI soil are higher than those at the G soil. CC and A treatments for SI had higher rates of N mineralization compared to FA, this values are also reflected in the treatments cumulative N values, making soils with any residual OM content have more available soil N. N Mineralization rates tend to be higher in soils with better aeration and clay content ( ), this supports the fact of SI rates being higher at earlier weeks due to having a loamy texture. G rates start rising on the 3rd week of incubation, rising from 15.8 mg N/kg soil to 24.1 mg N/kg soil, this show that up to date the G soil is capable of providing higher amounts of soil N compared to the Si soil starting at week 3. The AM treatment had a higher rates of N mineralization compared to FA, FA has values comparable to the SI values at week 4 but then surpasses SI rates. Data shows the cowpea amendment starts being mineralized the third week post amendment making it available as soil Nmin. This is also seen in the SI soil in which the AM treatment starts surpassing the CC treatment at a lower rate at week 3. Over all rates until now show the AM treatment as an efficient way to provide soil Nmin, which starts being available for the third week onward. Chart 3. N Mineralization Rates for Guayama and Santa Isabel 45 Nmin (mg N/kg) 40 35 G-F G-A SI-F SI-A SI-CC 30 25 20 15 10 5 0 0 1 2 3 4 5 Time (weeks) 34 6 7 The FA treatment was the less efficient in affording soil Nmin for both of the treated soils. In contrast the AM treatment was the most efficient Nmin provider for both soils. Accumulated N goes in order G-AM, G-FA, SI-AM, SI-CC, and SI-FA each with values of 58, 48.6, 39.2, 35.2, 25.3, mg N/kg, respectively. The G soil is able to provide more soil N in comparison to SI soil, this could be due to having a higher content of clay, therefore creating more desirable conditions for mineralization. In the G soil, A had around 10 mg N/kg soil more than FA revealing AM as a more efficient treatment. When referring to SI, AM provided around 4 mg N/kg soil more than CC and 14 mg N/kg soil more than Fa. CC rates showed a good input of Nmin at early stages making it a fair soil N provider, but cumulative values are lower than AM. It is expected for cumulative values on the AM treatment to keep being the highest among all treatments for both soils, followed by CC in SI and FA. Chart 4. Cumulative Inorganic Soil N 70.0 58.018 N Total (mg/kg soil) 60.0 50.0 48.606 39.214 40.0 30.0 Guayama FA Guayama AM Santa Isabel FA Santa Isabel AM Santa Isabel CC 35.279 25.329 20.0 10.0 0.0 Treatments Conclusions Soils amended with cowpea (AM) or with a history of cowpea rotation (CC) had a higher N accumulation of inorganic N (Nmin) at 6 weeks than unamended soils. The Jacaguas (Santa Isabel) soil with AM or CC treatments had higher rates of N mineralization during the early stages (0 to 3 weeks) but lower cumulative Nmin at 6 weeks than Guamaní (Guayama) soil. Cumulative Nmin at 6 weeks was in the order of Guamaní (AM) > Guamaní (F) > Jacaguas (AM) = Jacaguas CC > Jacaguas (F), with values of 58, 48.6, 39.2, 35.2, 25.3, mg N/kg, respectively. The results gathered to date suggest that amending soils with a covercrop residue such as cowpea is an efficient way to provide soil N to a crop and optimize the use of N fertilizers. It is expected for the AM treatment to keep having the highest M rate constant up to the 20 week and the higher cumulative N. Therefore confirming a CC amendment is the most efficient treatment for providing soil N. 35 Cited Literature Cabrera, M. L. (2007). Mineralización y Nitrificación: Procesos Claves en el Ciclo del Nitrógeno. Informaciones Agrónomicas de Cono Sur, #34, 1-9 Cabrera, M. L., Kissel, D.E., & Virgil M.F. (2005). Nitrogen mineralization from organic residues: Research opportunities. Journal of Enviromental Quality, #34, 75-79 Celaya-Michael, H., & Castellanos, A. E. (2011). Mineralización de Nitrógeno en el Suelo de Zonas Aridas y Semiáridas. Terra Latinoamericana, #29, 343-356 Dou, Z., Toth, J.D., Jabro, J.D., Fox, R.H., & Fritton, D.D. Soil Nitrogen Mineralization During Laboratory Incubation: Dynamics and Model Fitting. Soil Biology and Biochemistry, # 28, 625632 Garcia, F.O. (1992). Carnon and nitrogen dynamics and microbial ecology intallgrass prairie. (unedited Doctoral Tesis). Kansas State University, US. Kettler, T. A., Doran, J. W., & Gilbert, T. L. (2001). Simplified Method for Soil Particle-Size Determination to Accompany Soil-Quality Analyses. Soil Science of America, #65, 849-852 Kirschbaum M.U. 1994. The temperature dependence of soil organic matter decomposition, and the effect of global warming on soil organic storage. Soil Biol. Biochem. 27:753-760. Lacasta, C., Benitez, M., Maire, N., Meco, R. (2006). Efecto de la textura del suelo sobre diferentes parametros bioquímicos.VII Congreso SEAE Zaragoza 2006. #110 McDonald, N.T., Watson, C.J., Lalor, S.T.J., Laughlin, R.J., & Wall, D.P. (2013). Evaluation of soil tests for predicting nitrogen mineralization in temperate grassland soils. Soil Science Society of America Journal, #78, 1051-1064 Stanford, G., Carter, J.N., & Smith, S.J. (1974). Estimates of Potentialy Mineralizable Soil Nitrogen Based on Short-Term Incubations. Soil Science of America Proceedings, #38, 99-102 Stanford, G., Smith, S.J. (1972). Nitrogen mineralization potentials in soils. Soil Science of America Journal, #36, 465-472 36 Comparative Study of Organic and Conventional Farms in Puerto Rico Using Nematode Communities as Environmental Indicators Jennifer M. Rivera San Antonio and Roberto Vargas Department of Crops and Agro-Environmental Sciences Abstract The nematode communities provide us with valuable information that can be used as a mechanism to quantifying soil condition. By sorting nematode communities in conventional and organic farms we can determine the "environmental health" of the soil in both agricultural systems. The objective is to compare these systems in order to have a standard for “soil health” for specific crops and observe the effect of both agricultural systems in the soil nematode community. Two certified Organic farms in Puerto Rico (UPR Experimental Station, Lajas P.R. & Bananera Fabre, Sabana Grande P.R.) and one Conventional farm (Bananera Fabre, Sabana Grande P.R.) were selected for this study. Samples of 0.6kg, approximately, consisting of three subsamples, were isolated from each plot site and prepared for nematode extraction using the Christie & Perry Nematode extraction protocol with modifications and the Baermann technique. The Nematodes were classified according to their trophic level: bacterial feeding (BF), fungal feeding (FF), plant feeding (PF), predator (P) and omnivore (O). Results showed that nematode population in an Organic Farming System had higher diversity of the trophic group (BF, FF, O and P) and presented a lower percentage of Plant Feeding nematodes when compared to a Conventional farming System. In a Conventional Farming System we observed a higher population of (PF) nematodes and a low diversity of the trophic community (BF, FF, O and P). Apparently, high population of Plant Feeding nematodes suppresses other trophic groups in soil. Keywords: Nematodes, Organic versus Conventional Farms, Trophic level, Environment, Soil Resumen Las comunidades de nematodos proveen información valiosa que puede ser utilizada como mecanismos para clasificar la condición o calidad del suelo. Al clasificar las comunidades de nematodos en las fincas convencionales y orgánicas podemos determinar la "salud ambiental" del suelo en ambos sistemas agrícolas. El objetivo es comparar estos sistemas mediante un análisis de nematodos del suelo con el fin de obtener un estándar para la "salud del suelo" para cultivos específicos y observar el impacto de los sistemas agrícolas en la comunidad del suelo. Dos fincas Orgánicas certificadas en Puerto Rico (Estación Experimental de la UPR, Lajas PR y Bananera Fabre, Sabana Grande PR) y una finca convencional (Bananera Fabre, Sabana Grande PR) se seleccionaron para este estudio. Muestras de aproximadamente 0.6 kg, compuestas de tres sub-muestras, se colectaron y prepararon para la extracción de nematodos utilizando el protocolo de Christie y Perry modificado y la técnica de Baermann. Los nematodos fueron clasificados de acuerdo a los niveles tróficos: Bacteriófagos (BF), Fungívoros (FF), Fito-parasíticos (PF), Depredadores (P) y Omnívoros (O). La población de nematodos en un sistema de agricultura orgánica mostró una mayor diversidad en los niveles tróficos (BF, FF, O y P) y presentó un menor porcentaje de nematodos Fito-parasíticos. Aparentemente, una alta población de nematodos Fito-parasíticos suprimieron los restantes niveles tróficos. Palabras Claves: Nematodos, Finca Orgánica versus Convencional, Nivel trófico, Suelo 37 Introduction The origin of the word nematode comes from the Greek Laguage nema- meaning “thread” and ode meaning “like”, thread like. Nematodes are amongst the most abundant group of animals to exist. They range from 50μm diameter and 1mm in length. Nematode abundance in soils in managed and unmanaged (soils) ranges from 1–10 million individuals/m2 (Peterson and Luxton, 1982; Lavelle and Spain, 2001). The taxonomic classification of nematode constitute as the following; Kingdom: Animalia, Clade: Nematoida, Filum: Nematoda. Nematodes are recognized as a major consumer group in soils, generally grouped into four to five trophic categories based on the nature of their food, the structure of the stoma (mouth) and esophagus, and the method of feeding (Yeattes and Coleman, 1982). These groups are classified as bacterial feeders, fungal feeders, predatory feeders, omnivores, and plant feeders. These five feeding categories are based primarily on the stoma (Figure1). Figure 1. Nematodes can be classified into different feeding groups based on the structure of their mouthparts. (a) bacterial feeder, (b) fungal feeder, (c) plant feeder, (d) predator, (e) omnivore. Figure credit: Ed Zaborski, University of Illinois The nematode communities provide useful information that can be used as a soil quantifying mechanism (Berkelmans, et al., 2003). Nematodes live in a wide range of environments, while others live in a more restricted area. Scientists had been primarily based their studies of nematodes on the damage to crops but they also are essential organisms of the soils nutrient cycle and fertility. It has been proven that some nematodes are more responsive to resource enrichment than other organisms. (Ferris and Bongers, 2006). Bacterial-feeding nematodes contribute to the supply of nitrogen to the soil. By incorporating manure, compost or cover crops there it can stimulate bacterial growth and trigger an effect of the abundance of bacterial-feeding nematodes. This interaction can make nitrogen available for the plant intake. 38 Materials and Methods The study was conducted in a selection of three farms based upon their agricultural systems practices, one conventional farm and two organic farms. The Conventional Farm (Fabre) located in Sabana Grande, Puerto Rico produced bananas. The organic farms (Fabre) is also located in Sabana Grande, Puerto Rico and produced organic bananas in two stages: one 7 month stage and a 3 year stage project. Finally, the Organic farm of the Experimental Station of the University of Puerto Rico at Lajas which produced sweet peppers, pumpkin and eggplant. In all the farms the soil was classified as a Vertisol, Fraternity series. The Fraternity series are classified as fine, smectitic, isohyperthermic Typic Haplusters. These soils are moderately well drained with a mean annual temperature of 75 °F and a mean annual precipitation of 45 inches. These soils present a slow permeability. The soil samples consisted of a three sub-samples of approximately 0.6 kg. The Sub-samples were selected at a distance of (±1 feet) of the tree. The sample was placed in a plastic bag and kept in a cooler until processed. (Figure 2) Sample Collection at farms A total of 13 samples were collected from Organic Farm (5) and 8 samples of the same Conventional Farm due to the extended size of the farm. However, only the average value of the sample was taken under consideration as a representation of the effect of the conventional farming system. The nematodes were extracted using the Christie & Perry Nematode Extraction (Christie & Perry, 1951) with an adjustment in the process of breaking down the soil. A selection of 250cc of the soil and 250cc of water were placed in a blender in order to break down the soil aggregates. The blender was pulsed 2 to 4 times in order to obtain finer soil particles. This solution was transferred into a coarse sieve (#60) that contains about 20 to 24 meshes per inch to eliminate debris and a fine sieve (#360) to retain the nematodes. The samples were cleaned with water thoroughly until the water would pass through the sieve and would look clear. The suspension retained in the #360 sieve was transferred to a filter that was placed on a grid on top of a Baermann funnel. The funnels were placed in a rack and incubated for a period of 48 hrs 39 until collected in a vial. An aliquot of the sample was placed under the microscope for classification. The first 50 observed nematodes were taken under consideration and classified according to the trophic level. Results The nematode population in an Organic Farming System showed a higher diversity of the trophic group and presented a lower percentage of Plant Feeding nematodes when compared to a Conventional farming System. In the Organic Farming System (Bananas at a stage of a 7-month growth) we could observed the absence of Bacterial Feeding and Predator nematodes, whereas 5% of the observed nematodes were Fungal feeders, 32% Plant feeding nematodes and a 14% Omnivorous nematodes. In the same Organic Farm (Bananas at a stage of 3-Years) we could observed that the Plant Feeding nematode population decreased by 16% and the Omnivorous nematodes increased by a 35%. However we observed that the Predator nematode population disappeared. The Organic Farming System (5-Year Project) that produced Pumpkin, Eggplant and Sweet pepper showed a great diversity in the nematode population. We observed a decrease (67%) of Plant Feeding in soil influenced by Sweet peppers, compared to Pumpkin. In these samples, we also detected an increase in the population of Bacterial Feeding nematodes by 80%, Predator nematodes by 47% and Omnivorous nematodes by 40%. In these samples, the Fungal Feeding nematode population decreased by 37%. This effect can reflect the possible nematode population interaction that could be present once the Plant Feeding nematodes have decreased by more than a 50% of the original index. In the Conventional Farming System there was a population of 3% Bacterial Feeding nematodes, 1% of Fungal feeding nematodes, 36% of Plant feeding nematodes, 2% of Predator nematodes and an 8% of Omnivorous nematodes. By comparing both systems we observed that in the Organic Farming Systems the Plant Feeding nematode population decreased by a 22%. As an effect we observed an increase in Bacterial Feeding nematodes by a 33%, the Fungal Feeding nematodes by a 66%, the Predator nematodes by a 56% and the Omnivorous nematodes by 18%. Table 1. Results obtained form soil nematode extraction at Organic and Conventional Farming Systems. Bacterial Feeding Fungal Feeding Organic Banana 7-Month 0 0 Organic Banana 3-Year 0 Organic Pumpkin 5-Year Farm/Years Practice Trophic Level Plant Feeding Predator Omnivore 38 3 9 5 32 0 14 3 4 30 4 8 Organic Eggplant 5 Year 8 3 23 10 6 Organic Sweet Pepper 5-Year 16 3 10 8 13 Conventional Banana 12-Year 3 1 36 2 8 Population based on 50 nematodes counted on samples, Values were adjusted by 50. 40 Conclusion The impact of organic amendments to the soil decreases the plant feeding nematode population, allowing other trophic groups to prosper. In a Conventional Farming System of 12-years of practice the Plant Feeding nematodes suppress other trophic groups. By comparing a Conventional Farming System (12-years of practice) with an Organic Farming System (5-years of practice) a significant reduction of 22% in Plant Feeding nematodes could be reach as well as a more diversity in other nematode species. In a long-term organic system (3-years), it can promote the development of Omnivorous and Fungal Feeding nematodes. As a result, in an organic farming system we could support higher and more balanced diversity of the trophic groups. Cited Literature Berkelmans, R., Ferris, H., Tenuta, M. & van Bruggen, A.H.C. (2003). Effects of long-term crop management on nematode throphic levels other than plant feeders disappear after 1 year of disruptive soil management. Applied Soil ecology 23, 223-235. Bongers, T. (1990). The maturity index, an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14-19. Bongers, T. & Bongers, M. (1998). Functional diversity of nematodes. Appl. Soil Ecol. 10, 239251. Christie, J.R. & Perry, V.G. (1951). Removing nematodes from soil. Procedings of the Helminthological Society of Washington. 18, 106–108. Peterson, H., & Luxton, M. (1982). A comparative analysis of soil fauna populations and their role in decomposition processes. Oikos 39: 287–388. Ugarte, C. & Zaborski, E. (2014). Soil Nematodes in Organic Farming Systems Wilson, J.B. (1999). Guilds, functional types and ecological groups. Oikos. (86) 507-522. 41 Growth responses of lettuce plants (Lactuca sativa L., cv. “Black Seeded Simpson”) grown under a hydroponic system (NFT) receiving inorganic and organic nutrient solution. Mibet M. Cordero Ruiz and María del Carmen Librán Department of Crops and Agro Environmental Sciences Abstract There is a great concern and skepticism of people for the content of chemicals in foods consumed. Most of this concern came from people who already have a particular disease such as cancer, among others. Demand for crops grown under organic production systems began in Europe and has spread to, United States, Puerto Rico and other countries. For these reasons it is imperative to conduct research dealing with this issues. Today, lettuce crops (Lactuca sativa L.), is one of the most consumed and demanded horticultural crops in Puerto Rico. There is a need to conduct studies, to determine growth responses under an organic nutrient system as compared with an inorganic nutrient production system. The objectives of this research were; to determine the effectiveness of an organic fertilizer on lettuce plants grown in an NFT (Nutrient Film Technique) hydroponic system and to evaluate and compare the growth responses of these plants grown in an inorganic and organic nutrient solution. The lettuce cultivar to be evaluated is “Black Seeded Simpson”. During six months, two studies were conducted. In each study, two treatments were evaluated and replicated five times. Growth parameters evaluated were, number of leaves, height of the plant, and fresh weight. Results shown an optimum growth parameters on plants grown under an inorganic nutrient system as compared with plants grown in an organic solution nutrient system. Further studies should be conducted to evaluate the responses of crops grown under different organic fertilizers in hydroponic systems. Keywords: lettuce, NFT, organic fertilizer, inorganic fertilizer Resumen Existe una gran preocupación y escepticismo de las personas por el contenido de químicos en los alimentos que consumen. Mucha de las preocupaciones proviene de personas que ya tienen una enfermedad particular así como el cáncer, entre otras. La demanda por productos producidos bajo sistemas de producción orgánica comenzó en Europa y se ha extendido hacia los Estados Unidos, Puerto Rico y otros países. Por estas razones, es imperativo realizar investigación relacionada con esta problemática. Hoy día, el cultivo de la lechuga (Lactuca sativa L.), es uno de los cultivos hortícolas más consumidos y con mayor demanda en Puerto Rico. Existe una necesidad de realizar estudios, para determinar las respuestas de crecimiento bajo un sistema de producción nutritivo orgánico y compararlo con un sistema de producción nutritivo inorgánico. Los objetivos de esta investigación fueron: determinar la efectividad de un fertilizante orgánico en plantas de lechuga crecidas en un sistema hidropónico NFT (Nutrient Film Technique) y evaluar y comparar las respuestas de crecimiento de esas plantas crecidas en una solución mineral orgánica e inorgánica. El cultivar evaluado es la “Black Seeded Simpson”. Durante seis meses, dos estudios fueron realizados. En cada estudio, se evaluaron dos tratamientos con cinco réplicas cada uno. Los parámetros de crecimiento evaluados fueron, número de hojas, altura de 42 la planta, y peso fresco. Los resultados mostraron parámetros de crecimiento y rendimiento óptimo en las plantas crecidas en solución inorgánica al ser comparadas con las plantas crecidas bajo un sistema de solución nutritiva orgánica. Futuros estudios deben ser dirigidos a evaluar las respuestas de crecimiento de cultivos crecidos bajo diferentes fertilizantes orgánicos en un sistema hidropónico. Palabras claves: lechuga, NFT, abono orgánico, abono inorgánico Introduction In the last years the research results demonstrated that the demand for organic products in United States as well worldwide has been increasing significantly (Kortbech, 2002). Greens and herbs production is more through recirculating culture. Today, growers interest to provide crop nutrients from organic sources has increased (Kuack, 2013). There are many reasons of why the consumption and demand for organic products is increasing in different countries: United States 40.6%, Germany 12.7%, Japan 12.7%, France 6.3%, Italy 5.6%, Great Britain 4.6%, other Europe 16.2% and the Rest of the World 1.4%, these percentages represent the demand for organic vegetables over the inorganic vegetables (Willer, 2001). Parents, prefer the organic food for their children rather than food produced by conventional agriculture, in this manner they can prevent some diseases related with the chemicals used during farming. For this study organic fertilizer selected were, Seaweed Creme and Humega both manufactured by Bioflora®. Organic crops grown with organic solutions will not be added inorganic fertilizers or other chemical or synthetic substances that harms human health. Lettuce produced in this research are grown in a closed system NFT (Nutrient Film Technique) indicates that the plants are in contact with a sheet of mineral solution to a time interval. Crops grown in inorganic solutions are produced in a conventional hydroponic system, in which fertilizers and agrochemicals are used artificially synthesized. Materials and Methods This study was conducted in Finca Alzamora and Piñero Greenhouse, at University of Puerto Rico, Mayaguez Campus (Figure 1). Two studies were conducted. The first study were from September to December 2014 and the second study were from January to March 2015. Each study had two treatments replicated five times (Figure 2). In the first study plants were grown; T1- Inorganic nutrient solution with a Peters® soluble fertilizer 20-10-20/125 ppm N, T2Organic nutrient solution Seaweed Creme® fertilizer/soluble emulsion manufactured with marine algae. A second study was conducted; T1- Inorganic nutrient solution 20-10-20/150 ppm N, and T2- Organic nutrient solution Humega® (8% Humic Acid) (Figure 3). Lettuce seeds from cultivar Black Seeded Simpson were planted in a Clean Start Oasis® growing medium for hydroponics and placed into a germination tray (Figure 4). Plants were transplanted to an NFT hydroponic system with two inches height (Figure 5). Once they were transplanted to the four treatments electrical conductivity and pH, were monitored everyday by using an EC and pH meter (Figure 6). A daily inspection of lettuce plants watching the growth 43 and development of plants was performed. Once harvested lettuce we take data from growth parameters which are set numbers of leaves, plant height, pH, electrical conductivity and fresh weight (Figure 7). Once these data are taken we proceed to evaluate the results. Figure 1. Piñero Greenhouse Figure 2. First study (left) and second study (right). T1 inorganic (left) and T2 Organic (right) Figure 3. Organic (Seaweed Crème) and Inorganic fertilizer 44 Figure 4. Lettuce planted in a Clear Star Oasis Figure 5. Lettuce ready to be transplanted Figure 6. Electrical conductivity and pH meter Figure 7. Harvest time Results and Discussion First Study: September –December 2014 The first study was conducted at Finca Alzamora, Mayaguez Campus. Growth parameters results from the two treatments shown, that the average numbers of leaves grown in the organic treatment was 5 lower than plants as compared with the inorganic treatment which were 22 (Figure 8). In the organic treatment the lettuce plants did not have an acceptable growth and development as the plants in the inorganic treatment. The height of the lettuce plants in the organic treatment was 2 inches and in the inorganic treatment was 7.93 inches (Figure 9). The pH in the organic treatment was 7 and in the inorganic treatment were 6.6 (Figure 10). The electrical conductivity of the organic treatment was 0.35 mmhos and in the inorganic treatment 45 was 1.9 mmhos (Figure 11). This relevant difference in the electrical conductivity could be the reason of the poor growth and development of the lettuce plants grown in the organic treatment. The poor growth responses could be an effect of the low absorption by the plant from the nutrient in the organic fertilizer. According with Kuack, (2013) organic fertilizer could not be absorbed by the lettuce plants because were not degraded by the Nitrosomas bacteria and could not be absorbed easily. Figure 8 and 9. Number of leaves and plant height 46 Figure 10 and 11. pH and electrical conductivity Second Study: January – March 2015 The second study was conducted in Piñero greenhouse at Mayaguez Campus. The results of the two treatment, shown that in the organic treatment the average number of leaves were 50 meanwhile in the inorganic treatment were 5,000 (Figure 12). There is a considerable differences between the first study and the second study. Results shown that lettuce plants grown in 150 ppm of Nitrogen (20-10-20) shown higher growth parameters as compared with the first study. The reason should be due to an increase of the concentration of N, which affected on growth and development of the plants. The average height of the lettuce plants grown in the organic treatment was 2 inches and in the inorganic treatment was 11 inches (Figure 13). In the first study the results shown of the height of the lettuce plants in the organic treatment was 2 inches, showing equal results in this growth parameter. There are no considerable difference between both studies. The average fresh weight of the organic treatment was 8g as compared with the inorganic treatment which was 292g (Figure 14). The average pH solution of the organic treatment was 7.5 and in the inorganic treatment were 7.2 (Figure 15). The pH was not a factor 47 that affected the development of the lettuce plants in the organic treatment because the optimal pH in hydroponic nutrient solution is between 7.0 – 7.5 and the organic nutrient solution was on the range. The electrical conductivity of the organic treatment was 3 mmhos and in the inorganic treatment was 1.2 mmhos (Figure 16). This difference in the electrical conductivity could be the reason of the poor growth development of the lettuce plants in the organic treatment. The highest values of Electrical Conductivity could be the cause of leaves burnt of the plants in the organic nutrient solution. The values of EC (Electrical Conductivity) for a nutrient solution in hydroponic should be between 1.5 to 2.00 mmhos. Figures 12 and 13. Number of leaves and plant height 48 Figures 14, 15 and 16. pH in solution, electrical conductivity and fresh weight 49 Conclusion These results demonstrated in both studies that the organic nutrient solution containing organic fertilizers such as Seaweed Creme® and Humega®, did not stimulate an optimum growth and development in lettuce plants. Both studies shown similar results of growth parameters in treatments containing organic nutrient solution. Poor growth and development were observed. The lettuce plants from organic nutrient solution did not shown the expected development and quality as compared with the lettuce plants grown in the inorganic nutrient solution. Further studies should be conducted in manner to characterize and evaluate the chemical composition and plant responses produced with organic fertilizers used in hydroponic systems. Cited Literature Kuack, D. 2013. Using organic fertilizers for hydroponic lettuce production. University of Kansas. 2015. Willer, Helga and Y. Minov. 2001. Articulo elaborado con base en información extractada de “Organic Agriculture Worldwide”. 2015 50 Effects of Exogenous Biostimulants on Tahiti Lime (Citrus latifolia) Trees Affected by the Citrus Greening Disease in Lajas, Puerto Rico Alexandra I. Ramírez-Irizarry and J. Pablo Morales-Payan Department of Crops and Agro-Environmental Sciences Abstract Citrus greening is a relatively new disease in Puerto Rico, associated with the bacterium Candidatus Liberibacter asiaticus. The pathogen blocks the tree phloem, resulting general weakening of the trees, staggered death of branches, declining fruit yield, reduced fruit quality, and eventual death of the tree. It is currently considered the most devastating disease in citrus. There is no known cure for this disease, and its management relies on practices to contain its spread and to keep the tree alive and productive for as long as possible. The use of biostimulants (exogenous organic substances that accelerate growth and may enhance some physiological processes) may have potential in citrus greening management. Research was conducted in 2014-2015 in Lajas, Puerto Rico, to determine the effects of selected biostimulants on adult trees of Tahiti lime (Citrus latifolia) affected by the citrus greening disease. At the beginning of the experiment, plants were defoliated by approximately 75% and showed little vigor. Every 2 weeks we applied aqueous solutions of six biostimulants [(a) an extract of the alga Ascophyllum nodosum, (b) a formulation based on soluble silicon, (c) a blend of vitamins and enzymes, (d) gibberellin 4/7, (e) an extract of the plant Reynoutria sachalinensis, and (f) an extract of plants rich in brassinosteroids, spraying the leaves to run-off. All the biostimulants were applied with an organic surfactant. Thus, a group of plants was sprayed only with the surfactant dissolved in water to account for its potential effects on the trees. Also, plants without application of biostimulant or surfactant were evaluated as checks. There were eight trees per treatment, in a completely randomized design. The results of trees treated with the surfactant were the same as those of check trees. Chlorophyll concentration in the leaves was not affected by the biostimulants. As compared to check trees, Tahiti lime trees treated with gibberellin 4/7 tended to have larger crown diameters and were more vigorous than check plants. After four applications of the biostimulants, trees treated with the silicon biostimulant had a greater survival rate than those of other treatments. At the time of writing this abstract, this field research is still ongoing. Keywords: Citrus, Biostimulants, Citrus greening Resumen El citrus greening en una enfermedad relativamente nueva en Puerto Rico, asociada a la bacteria Candidatus Liberibacter asiaticus. El patógeno bloquea el floema del árbol, provocando su deterioro progresivo, reducción de su productividad y eventualmente su muerte. Actualmente se considera la enfermedad más devastadora de los cítricos, ya que no se conoce cura para esta enfermedad. Las prácticas usadas en huertos enfermos se enfocan en prolongar la vida de los árboles el mayor tiempo posible. El uso de bioestimulantes (sustancias orgánicas exógenas que aceleran el crecimiento y pueden mejorar algunos procesos fisiológicos) es una de las prácticas con potencial para retardar el deterioro de plantas afectadas por citrus greening. Cada 2 semanas se aplicaron soluciones acuosas de seis bioestimulantes [(a) un extracto de alga Ascophyllum nodosum, (b) una formulación basada en silicio soluble, (c) una mezcla de vitaminas y enzimas, 51 (d) geberelina 4/7 (e) extracto de la planta Reynoutria sachalinensis y (f) extracto de plantas ricas en brasinoesteroides (con un agente surfactante para ayudar a los bioestimulantes a penetrar a las hojas pulverizadas hasta el escurrimiento. Además, un grupo de plantas se pulverizadas solo con el surfactante disuelto en agua para saber sus posibles efectos en los arboles. También, las plantas sin aplicación de bioestimulantes o surfactante se evaluaron como controles. Hubo 8 arboles por tratamiento con un orden completamente al azar. Los resultados muestran que el surfactante por si mismo no afecto a los árboles tratados. No se encontró efecto significativo de los tratamientos en la concentración de clorofila. Los arboles tratados giberelina 4/7 tendieron a tener coronas (sistemas de ramas) de mayor diámetro y a ser más vigorosos que los árboles sin bioestimulante. Luego de cuatro aplicaciones de los tratamientos, los arboles tratados con la formulación de silicio tuvieron una tasa de supervivencia 25% mayor que los arboles sin bioestimulante. Al momento de redactar este resumen, la investigación continúa. Palabras Claves: Bioestimulantes, limón Tahití, citrus greening Introduction Citrus in Puerto Rico are traditionally fruits of economic importance, and they generally were among the top four fruits in terms of annual farm gate value. In Puerto Rico, common limes (Citrus aurantifolia) and Tahiti or Persian limes (Citrus latifolia) are among the favorite citrus, with more than 90% of the limes marketed in the island are imported. The number of new lime orchards planted in the first decade of the 21st century was on the rise in Puerto Rico, when a new disease, the Citrus greening, was detected in the island in 2009. It has been estimated that from 2009 and 2014 approximately 80% of the orchards have been affected by the disease, reducing their productivity and longevity. The Citrus greening disease has been associated with the bacterium Candidatus Liberobacter asiaticus, transmitted by the citrus psyllid Diaphoria citri. The psyllid causes direct damage by sucking sap from the tree, but this is considered minor or tolerable damage as compared to the indirect and permanent damage caused by its transmitting the pathogen that causes the Citrus greening into the tree while feeding from it. The transmitted bacterium stays in the phloem, eventually blocking it, which results in loss of vigor, poor growth, chlorotic mosaic (green and yellow) with irregular patterns in the leaves, branches with sparse foliage, branch die back, and loss of leaves, flowers and fruits. Fruits that do not abort may have abnormal coloration, are generally small, have low juice content and poor juice quality. In Puerto Rico, losses due to this disease vary, with some orchards being completely lost, while in other orchards 50% of the lime trees are known to have died between 2011 and 2014. In Florida, USA, the estimated loss to Citrus greening was $3,600 million between 2006 and 2012 (Evans et al., 2014). All the widely grown citrus varieties are susceptible to the disease, and there is no known cure for the Citrus greening. Until new resistant varieties with commercial value are developed, the disease is managed with practices to retard the infection and development of the disease, and to prolong the commercial life of the trees. Management practices tried in other countries include intercropping with guava (Psidium guajava) (which is said to repel the vector but has not been effective in most places), growing transplants in enclosed structures to ensure new plantings are 52 done with healthy trees, intensive programs of foliar fertilization and insecticides, and removing symptomatic citrus trees (Ichinose et al., 2012; Grafton-Cardwelll et al., 2013). Those practices have had varying degrees of success, but there is need for more research to explore other alternatives that may prolong the commercial life of infected trees. Biostimulants are organic substances that in relatively small concentrations may influence the physiology of plants, resulting in accelerated growth, increased uptake and transport of essential nutrients, enhanced photosynthetic activity, and increased tolerance to biotic and/or abiotic stress, which may lead to reduced flower and fruit abortion, and higher yield and quality (De la Cruz, 2013). A number of biostimulants are approved for organic systems, offering a means for conventional and organic growers to enhance crop performance with little environmental impact (Flores-Torres, 2013). The effects of biostimulants in trees affected by the Citrus greening are unknown. Infected trees will eventually die, but it is possible that because of their effects on growth and vigor bioestimulants may help prolong the commercial life or trees with the citrus greening. The use of organic biostimulants in lime trees affected by the citrus greening may be a promising practice, whose research we are pioneering in Puerto Rico. The objective of this research was to determine the effects of selected foliar-applied biostimulants on the growth and survival of organicallygrown Tahiti lime trees affected by the citrus greening in Puerto Rico. Materials and Methods This field experiment took place in the Agricultural Experiment Station in Lajas, Puerto Rico (Figure 1). The trees used are Tahiti lime grafted onto in ‘Cleopatra’ tangerine (Citrus reticulata), planted in 2006. The orchard has been grown organically since 2007. Trees started to show symptoms of greening in late 2012. Figure 1. View of the experimental orchard from above. Lajas, Puerto Rico. 53 We tested six biostimulants (Table 1), which included a seaweed (Ascophyllum nodosum) extract; a formulation of soluble silicon; a blend of brassins, enzymes and vitamins; a plant extract rich in natural brassinosteroids; an extract of the plant Reynoutria sachalinensis; and gibberellin 4/7. All of these biostimulants have been known to enhance citrus performance in the absence of citrus greening (Diaz-Candelas, 2013), and are approved for use in organic systems. The rates used (Table 1) were those recommended by the manufacturers for use in citrus. All the bioestimulants were applied with an organic surfactant (an extract of yucca and garlic), and a treatment with surfactant alone in water was used to test potential effects of the surfactant on the plant. An untreated check (no surfactant, no biostimulant) was also included in the experiment. The biostimulants were applied in aqueous solutions, spraying all the foliage to run-off (approximately 500 ml per tree), at a frequency of 14 days. Table 1. Description of treatments tested on citrus greening-affected Tahiti lime trees in Lajas, Puerto Rico, 2014-2015. Treatment Active ingredients Commercial Formulation Rate of commercial formulation per liter of water Surfactant Yucca spp. and garlic (Allium sativum) extract BioLink ® 1 ml Ascophyllum nodosum seaweed extract Citokininins, gibberellin, oligosaccharides, amino acids and betaine Stimplex® 5 ml Natural extract Vitamins, brassins and enzymes Organic Vitazyme® 5 ml Blend of organic acid and nutrients Blend of silicon with humic and fulvic acids and micronutrients QuickSol® 2.5 ml Gibberella fujikuroi fungi derived Giberelins 4/7 ProVide® 50 mg of active ingredient Plant extract Brassins Comcat® 1g Reynoutria sachalinensis plant extract Undisclosed Regalia® 5 ml Control 54 Mentioning commercial names does not imply a special endorsement from the authors or from the University to that specific formulation or manufacturer; it is done solely to specify the formulation used in the experiment as it was available at the time of conducting the research. We used a completely randomized design, with eight trees per treatment. Canopy height and diameter, number of symptomatic and asymptomatic branches, SPAD values in expanded leaves (associated with chlorophyll concentration), vigor (a 0-5 visual scale modified from Inch (2014) where 0 is dead and 5 is an asymptomatic tree), and tree survival rate were evaluated. The results were submitted to analysis of variance and separation of means as appropriate. % Increase above check tress Results and Discussion There was no effect of surfactant alone on plant growth and survival. None of the treatments significantly affected the SPAD values of expanded leaves, indicating that on average leaf coloration (a reflection of chlorophyll concentration) was not statistically different (data not shown). It is possible that the disease affected the availability of nutrients necessary to synthesize and/or maintain chlorophyll molecules, therefore preventing biostimulants from increasing chlorophyll concentration as they have been known to do in healthy fruit crop trees. Flowering and fruit set varied considerably between trees, and yield will be determined at harvest. As compared to check trees, Tahiti lime trees treated with gibberellin 4/7 tended to have larger crown height and diameter (Figure 2); this result may be explained by the effect of gibberellins on promotion of vegetative elongation, particularly on stems and branches. Tree vigor was also greater in plants treated with gibberellin 4/7 (an average value of 2.4 in the 0-5 scale) than in check trees (an average value of 1.33 in the 0-5 scale). Asymptomatic new branches tended to be more abundant in trees treated with biostimulants than in check trees. 25 Gibberellin 15 11 Vitamin/enzyme/brassin Silicon 0 Reynoutria extract Figure 2. Effect of selected biostimulants on crown diameter (% above check plants) in Tahiti lime trees affected by citrus greening. Lajas, 2015. 55 After four applications of the biostimulants, tree survival was 100% in trees treated with the silicon formulation, as compared to approximately 75% survival in check plants and those treated with the Reynoutria sachalinensis formulation, and approximately 85% in all the other treatments (Figure 3). These results may be attributed to the growth promoting properties of the active ingredients (gibberellins, citokinins, amino acids, brassins, vitamins, enzymes) in some of the biostimulants tested in this research, which may have allowed the plants to continue growing sufficiently to survive despite the disease. Figure 3. Survival of Tahiti lime trees with citrus greening as affected by selected biostimulants. Conclusions As of March 2015, the experiment is still being conducted. So far, some biostimulants appear to have an effect on tree survival and growth. While fruit yield has not been evaluated, effects on growth and survival may be an indicative that some of the treatments tested in this research may be helpful in prolonging the life of citrus greening-affected Tahiti lime trees grown organically. Cited Literature Evans, E. A., Ballen, F. H., & Crane, J. H. 2014. Economic Potential of Producing Tahiti Limes in Southern Florida in the Presence of Citrus Canker and Citrus Greening. HortTechnology, 24:99-106. 56 De La Cruz-Rodríguez, L. F. 2013. Evaluación de dos bioestimulantes agrícolas en naranja dulce (Citrus sinensis) ‘Valencia Tardía’ en la región montañosa centro-norte de Puerto Rico. Tesis de Maestría en Horticultura. Universidad de Puerto Rico, Recinto de Mayaguez.p.4-13. Díaz-Candelas, C. C. 2013. Effect of exogenous bio-regulators on organically-managed Tahiti lime (Citrus latifolia Tanaka) fruit and essential oil productivity and quality. Tesis de Maestría en Horticultura. Universidad de Puerto Rico, Recinto de Mayaguez.p.3-17. Grafton-Cardwell, E. E., Stelinski, L. L., & Stansly, P. A. 2013. Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Annual review of entomology 58:413-432. Flores-Torres, C. R. 2013. Efectos de bioestimulantes para sistemas orgánicos y sustentables en limón ‘Tahití’ (Citrus latifolia Tanaka). Tesis de Maestría en Horticultura. Universidad de Puerto Rico, Recinto de Mayaguez.p.5-19. Ichinose, K., Hoa, N. V., Bang, D. V., Tuan, D. H., & Dien, L. Q. 2012. Limited efficacy of guava interplanting on citrus greening disease: Effectiveness of protection against disease invasion breaks down after one year. Crop Protection, 34, 119-126. Inch, S. 2014. Evaluation of Commercial Citrus Cultivars for Field Tolerance/Resistance to Huanglongbing in East Central Florida. Abstracts of the American Society for horticulture Meeting, Orlando, Florida. July 2014. 57 Rhytismatales and other pathogenic fungal species of native trees of Puerto Rico Verónica M. Rivera Vega1 and Lydia. I. Rivera Vargas2 1 Undergraduate student, General Agriculture Program, 2Professor, Department of Crops and Agro-Environmental Sciences, University of Puerto Rico-Mayaguez Campus, Mayaguez, PR 00681 e-mail: [email protected]; [email protected] Abstract Most forests in Puerto Rico are devoted to the preservation of natural areas and watersheds, to the conservation of biodiversity, and as such, as a source of passive recreation. Puerto Rico’s flora includes 3,243 plant species, and at close to 1,050 are trees, estimating endemism to be around 13.6%. Currently both biotic and abiotic factors are identified as clear and present threats to this biological treasure. Among them, the advent of biological invasions by exotic pests and diseases. Our research objective was to identify and characterize microorganisms affecting native tree species. Samples were collected from tree species showing diseased tissues from different locations and ecosystems around the island. Locations sampled were: the University of Puerto Rico-Mayaguez Campus, the “Arboretum Metropolitano del Parque Doña Inés” in Tujillo Alto, and coastal areas at Joyuda, Cabo Rojo, Puerto Rico. Free-hand sections of plant tissue containing fungal reproductive structures were examined by light and scanning electron microscopy. To stimulate sporulation of pathogenic fungi, moist chambers containing infected plant tissues were prepared. In addition, fungi were isolated from tissue sections after placing them in culture media. For some specimens, DNA was extracted, sequenced and analyzed. Five different species of fungi were identified on five endemic and native trees of Puerto Rico. A fungal specimen belonging to the Order Rhytismatales was identified on the leaves of Cocoloba nervosa collected at the “Arboretrum Metropolitano del Parque Doña Ines”. Another fungus, Meliola spp. was identified on leaves of Crescentia cujete collected at the same location. Through the use of molecular techniques, we confirmed the phytopathogenic fungus, Hilberina caudata affecting the leaves of Cordia sebestena at the coastal areas of Joyuda, Cabo Rojo, Puerto Rico. As part of this project we also identified the parasitic green algae, Cephaleuros virescens in Simarouba talae tree. Puerto Rico is an island with high density population and fragile ecosystems. Thus understanding fungal diseases affecting our flora will allow us to develop an effective plan for the conservation of native species of trees in Puerto Rico and the Caribbean. Key words: Rhytismatales, phytopathogenic fungi, native trees. Resumen La mayoría de los bosques en Puerto Rico están dedicados a la preservación de los espacios naturales y cuencas hidrográficas, y también para la conservación de la biodiversidad, y como tal, como una fuente de recreación pasiva. La flora de Puerto Rico incluye 3,243 especies de plantas, y cerca de 1,050 árboles, la estimación de endemismo es alrededor de un 13.6%. Actualmente tanto los factores bióticos y abióticos se identifican como amenazas claras y presentes a este tesoro biológico. Entre ellos, la llegada de invasiones biológicas de plagas exóticas y enfermedades. El objetivo de nuestra investigación fue identificar y caracterizar los microorganismos que afectan a las especies de árboles nativos. Se 58 recogieron muestras de las especies de árboles que mostraban tejidos enfermos de diferentes lugares y ecosistemas de la isla. Las ubicaciones donde se recolectaron las muestreas fueron: la Universidad de Puerto Rico Recinto de Mayagüez, el Arboretum Metropolitano del Parque Doña Inés en Tujillo Alto y las zonas costeras en Joyuda, Cabo Rojo, Puerto Rico. Cortes histológicos a mano libre del tejido vegetal, que contenían estructuras reproductivas de los hongos, fueron examinadas por microscopía de luz y electrónica de rastreo. Para estimular la esporulación de hongos patógenos, se prepararon cámaras húmedas con tejidos de las plantas infectadas. En adicción, los hongos fueron aislados de secciones de tejido vegetal luego de, haber sido colocados a crecer en medios de cultivo. Para algunas muestras, se extrajo ADN y este fue secuenciado y analizado. Se identificaron cinco especies diferentes de hongos en cinco árboles endémicos y nativos de Puerto Rico. Una muestra de hongos pertenecientes a la Orden Rhytismatales fue identificado en las hojas de Cocoloba nervosa recogidos en el Arboretrum Metropolitano del Parque Doña Inés. Otro hongo, Meliola spp. fue identificado en las hojas de Crescentia cujete recogido en el mismo lugar. A través del uso de técnicas moleculares, se confirmó que el hongo fitopatógeno, Hilberina caudata afecta a las hojas de Cordia sebestena en las zonas costeras de Joyuda, Cabo Rojo, Puerto Rico. Como parte de este proyecto también identificamos las algas verdes parasitaria, Cephaleuros virescens en el árbol Simarouba talae. Puerto Rico es una isla con gran densidad de población y frágiles ecosistemas. Por ende, la comprensión de las enfermedades fúngicas que afectan a nuestra flora nos permitirá desarrollar un plan efectivo para la conservación de las especies nativas de árboles en Puerto Rico y el Caribe. Palabras claves: Rhytismatales, hongos fitopatógenos, arboles nativos Introduction Since ancient times trees have been important sources of income and fuel. At the present, there is no organized production of hardwoods in Puerto Rico, with limited economic activity in the form of artisanal charcoal and precious wood production. Most forests in Puerto Rico are devoted to the preservation of natural areas and watersheds, to the conservation of biodiversity, and as such, as a source of passive recreation. According to Acevedo-Rodríguez and Strong (2012) the flora of Puerto Rico includes 3,243 plant species, and at close to 1,050 are trees. These authors also estimate endemism to be around 13.6%. Currently both biotic and abiotic factors are identified as clear and present threats to this biological treasure. Among them are the encroachment by human settlements, and lately, the advent of biological invasions by exotic pests and diseases. The study of diseases affecting tree species in Puerto Rico is almost not existent. Almost plant pathology scientific work has been done for agricultural plants, so a great knowledge void exists on threats to forest species, especially those affecting native and endemic plants. Thus, the principal objective of this investigation was to begin a process of initial identification of fungal microorganisms that affect native and endemic species of trees in Puerto Rico. 59 Material and Methods Leaf and bark samples collection: Leaf and bark tissue samples from endemic tree species were collected at: the University of Puerto Rico-Mayaguez Campus (UPRM), the “Arboretum Metropolitano del Parque Doña Inés” in Tujillo Alto, and coastal areas at Joyuda, Cabo Rojo, Puerto Rico. Six species of trees were selected: Cocoloba nervosa, Cordia sebestena, Crescentia cujete, Juglans jamaicensis, Simarouba talae and an unknown species. These species showed symptoms of infection characterized by: wilting, presence of necrotic tissues, and tar spots or green islands on leaves. Bark was collected from Juglans jamaicensis and from a fallen unknown tree at the UPRM who’s trunk and bark showed signs of putrescence. Samples were placed in individual plastic bags in a portable cooler and transported to the Plant Pathology Lab at UPR-Mayaguez. Samples were kept at 10°C before processing. Microorganism Identification: For microorganism’s identification, reproductive structures were mounted on microscope slides using a drop of lactophenol and examined with a light microscope (Olympus, Ontario, Canada). For fungal or oomycetes specimens, tissue sections of about 4mm were dissected from an area close to signs or spots. Dissected sections were transferred to two culture media: potato dextrose agar (i.e. PDA) for fungal isolation and PARPH agar for oomycetes isolation. PARPH Agar contained cornmeal and antibiotics such as: pimaricin (5mg), rifampicin (10mg), ampicillin (250mg), hymezaxol (50mg) and pentachloronitrobenzene (100mg). Petri plates were incubated at 26̊C. Once growth was observed on Petri plates, colonies were purified for identification. Taxonomic keys were used for fungal identification (Barnett and Hunter, 1982; Hanlin, 1990). In addition, to achieve fungal sporulation, moist chambers were prepared from plant tissue and incubated at room temperature. Direct isolation from fungal reproductive structures was used for further purification and identification. Scanning electron microscopy (SEM) was used to further examined samples from leaves of C. nervosa and C. sebestena. Tissue sections (approx. 7 mm) were dehydrated and processed previous to examination following the methodology stipulated by Bonilla (2012). For fungal DNA analysis, DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Alemania), following manufacturer’s instructions. DNA samples were amplified by PCR in a thermocycler (Eppendorf, Hauppauge, NY). Reaction mix consisted of: 10µL of AmpliTaq Gold® Fast PCR master mix (Life Technologies, Carlsbad, CA), 1.5µL of each primer, 3µL of molecular water and 4µL from sample DNA. Primer sets used were commonly employed to identify fungi belonging within the Order Rhytismatales. Primer sets were: 5.8SR/LR6, mrSSU1/mrSSU3R, LROR/LR6 and LR3/LR6 (Lantz et al., 2011; Vilgalys and Hester, 1990). In addition primer set ITS1/ITS4 were used (White et al., 1990). Thirty PCR cycles were performed using the following parameters: 94°C denaturation step (1 min), 50°C annealing step (45 s), 50 to 72°C ramp (1 min), and primer extension at 72°C (1 min). A final 7-min incubation step at 72°C was added after the final cycle. PCR products were visualized in 1.2% agarose gel electrophoresis containing TAE buffer and 2.5µL of ethidium bromide. Molecular marker or ladder used was100bp (DNA 60 Ladder, BioLabs, New England). PCR product was purified by QI Aquick® Gel Extraction Kit, (Qiagen, Germantown, MD) following manufactures instructions. DNA was sequenced at commercial facilities (Macrogen Inc., Rockvillle, MD). Nucleotide sequences were analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Results and Discussion Three phytopathogenic fungi were identified on leaves of C. nervosa, C. sebestena and C, cujete. Two saprophytic fungi were identified from bark of J. jamaicensis and from an unknown fallen tree (Table 1). Table 1 summarized tree species and tissue sampled, and microorganisms identified. On leaves of C. cujete, we observed pigmented mycelia, hyphopodia and ascospores of the phytopathogenic fungus, Meliola sp. using light microscopy (Figure 1). On leaves of C. nervosa, we observed reproductive structures of Melasmia sp., the imperfect state a fungus belonging to the Order Rhytismatales, which is also a phytopathogenic fungus (Figure 2). On leaves of C. sebestena we observed ascospores of Hilberina caudata.using light microscopy (Figure 3). Through molecular characterization of the LSS region, we were able to confirm the identity of the pythopathogenic fungus as H. caudata (Figure 4). Table 1. Trees species and tissue sampled, location and microorganism identified. Tree Location Coccoloba nervosa Parque Doña Inés Cordia sebestena Crescentia cujete Juglans jamaicensis Simarouba talae Unknown #1 Joyuda , Cabo Rojo Parque Doña Inés Parque Doña Inés Parque Doña Inés UPRM Tissue Sample Leaf Type of Microorganism Phytopathogenic Fungi Leaf Microorganism Identified Melasmia sp. Imperfect state, Order Rhytismatales Hilberina caudata Leaf Meliola sp. Phytopathogenic Fungi Bark Order Hysteriales Saprophytic Fungi Leaf Cephaleuros virescens Trichoderma sp. Plant parasitic green algae Saprophytic Fungi Bark Phytopathogenic Fungi Only samples from C. nervosa and C. sebestena were submitted to DNA analysis. We were unable to obtained PCR products using primer sets ITS1/ITS4 and 5.8R/LR6. A 250 bp PCR product were obtained using a primer set of a mitochondrial region: mrSSU1/mrSSU3R but DNA sequences were too short to make final conclusions (data not shown). Successful PCR amplification was obtained using primer set LROR/LR6 for C. 61 sebestena. A final product of 1,165pb was amplified (Figure 4). The sequence obtained showed 87% of homology to H. caudata when analyzed by BLAST. A saprophytic fungus, Trichoderma sp. was isolated from bark of a fallen unknown tree. Another saprophytic fungus was identified by direct examination of bark tissue of J. jamaicensis, the fungus belongs to the Order Hysteriales. No oomycete were isolated from vegetative tissue. Thus, PDA was better isolation media than PARPH. As part of this project we also identified the parasitic green algae, Cephaleuros virescens in Simarouba talae tree (Figure 5). A B p h Figure 1. Crescentia cujete: A. Sooty mold growing on leaf; B. Reproductive structures of the phytopatogenic fungus, Meliola sp. examined by light microscopy (40x). Mycelium, perithecia, (p) and mucronate hyphopodia (h) were observed. Insert: Multicellular ascospores. A B D C E F Figure 2: Cocoloba nervosa: A: Leaf showing tar spot and chlorosis. B: Group of pycnidia causing spots on leaf. C: Conidias of Melasmia sp. (40X). D: Unknown structures observed on imperfect state, Melasmia sp. (40X). E. Conidiophores (40X). F: Mycelial growth protruding through leaf stomata observed by Scanning Electron Microscopy. 62 A A B C Figure 3. Cordia sebestena: A. Green islands on leaf (arrow). Photo by Alejandro Segarra; B. Ascospores of Hilberina caudata. (40X); C. Mycelial growth on leaf observed by Scanning Electron Microscopy. C 63 L 1 2 3 4 1 2 3 4 C 1,165pb Figure 4: Agarose gel of PCR products using primer set LR3/LR6 (black numbers) and LROR/LR6 (whire numbers) for samples of C. sebestena (1 and 2) and C. nervosa (3 and 4; arrow). L= ladder; C = Control water. A B Figure 5. Simarouba talae A. Leaf showing green algae spots. Photo by Victor Gonzalez. B. Cepahleuros virescens (Photo from APS Press) 64 Symptoms observed on leaves of C. nervosa were similar to tar spots. A free-hand histological section of leaf tissue and direct examination by light microcopy allowed the identification of Melasmia sp., the imperfect stage of a fungus belonging to the order Rhytismatales (Figure 2). We observed reproductive structures such as pycnidia (Figure 2D), and superficial mycelium invading leaves and protruding through stomata (Figure 2F), conidiophores were simple or branched (Fig.2E) and conidias hyaline and allantoid (Fig.2C) (Barnett and Hunter, 1998). Green islands were observed on leaves of C. sebestena. The fungus invaded the stomata. Ascospores were clavate, unicellular and hyaline, with the basal end bent (Figure 3B) (Miller et al., 2014). Through molecular analysis we obtained and identification with 87% of homology with the phytopathogenic fungus Hilberina caudata (Ascomycota). On leaves of Crescentia cujete, we identified the fungus Meliola sp.. This genus is abundant in the tropics and is known for the production of abundant superficial dark brown septate mycelium. Mucronate hyphopodia are commonly observed associated with mycelium. Globose, ostiolate perithecia are produced superficially on leaves containing multicellular dark brown ascospores (Fig.1) (Kenneth, 2008). Trichoderma sp., a saprophytic fungus, was recollected from an unknown fallen tree that was at the stage of decay. This fungus is commonly found in different climate zones, having numerous means of survival and proliferate (Harman, G and Kubicek, 2005). Juglans jamaicensis was also found at the stage of decay at moment of the recollection, identifying the saprophytic fungus from Order Hysteriales. We were unable to identify the pathogenic organism that caused its death. Simarouba talae is a perennial tree, native from P.R that grows on the mountains of the island. On leaves of S. talae we identified C. virescens a green algae that affect mostly trees of tropical and subtropical zones. This algae is commonly found affecting mango leaves (Fig.2) (Litz, 2009). This algae requires high relative humidity to caused infections. Initially it produces a spot of about 5 to 8mm of diameter, with a red to orange coloration turning to a necrotic lesion with age (Litz, 2009). Conclusion In conclusion, we identified five different species of fungi in five endemic and native trees of Puerto Rico. Three of them, H. caudata, Meliola sp. and Melasmia sp. are known phytopathogenic species. Trichoderma sp. and a fungus from the Order Hysteriales are saprophytic species. A green algae, C. virescens was identified on leaves of a native tree of P.R. Puerto Rico is an island with high density population and fragile ecosystems. Thus understanding fungal diseases affecting our flora will allow us to develop an effective plan for the conservation of natives and endemic species of trees in Puerto Rico and the Caribbean. Cited Literature Acevedo-Rodríguez, P., Strong, M. T. 2012. Catalogue of seed plants of the West Indies. Smithsonian Contributions to Botany. 98: 1-1192. 65 Barnett, H.L. and Hunter, B.B. 1998. 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The Mycological Society of America, KS, USA Litz, R.E. 2009. The Mango: Botany, production and uses, 2nd edition. CAB International. MA, USA, 232-233pp. Miller, A.M., S.M. Huhndorf, and J. Fournier. 2014. Phylogenetic relationships of five uncommon species of Lasiosphaeria and three new species in the Helminthosphaeriaceae (Sordariomycetes). Mycologia, 106(3), 2014, pp. 505–524 Vilgalys R. and Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246. Acknowledgements This project was supported by CARIPAC program and Agriculture and Food Research Initiative Competitive Grant no. 2012-01871 from the USDA National Institute of Food and Agriculture, Hispanic Serving Institution. 66 “This project was supported by the Resident Instruction in Insular Areas Program Competitive Grant no. 201370004-21018 and 2014-70004-22033 from the USDA National Institute of Food and Agriculture.”