Implantação de planos de fogo controlado na gestão dos recursos alimentares para cavalos da raça Garrana em Valença

Dissertação apresentada à Escola Superior Agrária do Instituto Politécnico de Castelo Branco para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Tecnologias e Sustentabilidade dos Sistema Florestais.

Aplicación de técnicas de PCR en tiempo real a la trazabilidad y autenticidad de los piensos

La trazabilidad y el correcto etiquetado de los piensos y sus ingredientes son factores esenciales para prevenir fraudes y garantizar la seguridad alimentaria. En el ámbito de la lucha contra las Encefalopatías Espongiformes Transmisibles (EETs), la prohibición de la Unión Europea (UE) de alimentar a rumiantes y otros animales de granja con harinas de carne y huesos derivadas de animales, hace necesaria la disponibilidad de metodologías que permitan identificar el origen de las materias primas e ingredientes presentes en los piensos. El método oficial de análisis microscópico tradicionalmente empleado para este fin presenta limitaciones a la hora de diferenciar entre los huesos de mamíferos y de aves, así como para determinar el origen animal específico de las partículas detectadas. Por ello, una de las prioridades de la UE en los últimos años ha sido potenciar la búsqueda y desarrollo de técnicas analíticas alternativas que permitan la detección específica de todos los componentes que integran los piensos. Teniendo en cuenta estos aspectos, en esta Tesis Doctoral se han desarrollado técnicas de PCR en tiempo real con sondas TaqMan® para el control de autenticidad y trazabilidad de ingredientes de origen animal utilizados en la fabricación de los piensos. Las especies objeto de este trabajo han sido: vaca (Bos taurus), oveja (Ovis aries), cabra (Capra hircos), grupo rumiante, cerdo (Sus scrofa), pollo (Gallus gallos), pavo (Meleagris g-allopavo), pato (Anal platyrhynchos x Cairina moschata), oca (Anser anser), grupo aviar, caballo (Equus caballus), conejo (Oryctolagus cuniculus), liebre (Lepus capensis), grupo lepórido (conejo y liebre) y pescados...

Avaliação da viabilidade da aplicação de técnicas biomoleculares na autenticação de produtos alimentares

A carne continua a ser a fonte proteica mais comum no quotidiano das pessoas. Além disso, os produtos cárneos processados apresentam-se como uma mais-valia nas suas vidas agitadas. Este tipo de produto torna difícil a diferenciação das carnes utilizadas na sua confecção, sendo por isso propícios a adulteração. A Reacção em Cadeia da Polimerase (PCR) tem ganho cada vez mais importância nos laboratórios de biologia molecular, revelando-se uma técnica de análise rápida, sensível e altamente específica na identificação de espécies em produtos alimentares. No entanto, vários factores podem interferir com o processo de amplificação, pelo que alguns cuidados devem ser implementados desde a aquisição da amostra a analisar, ao seu acondicionamento e posterior extração de ADN. Existem inúmeros protocolos de extração de ADN, devendo para cada estudo avaliar-se e optar-se pelo mais adequado, considerando a finalidade estabelecida para a amostra extraída. O trabalho laboratorial apresentado nesta dissertação baseou-se em três etapas principais. Inicialmente, avaliaram-se diferentes protocolos de extração de ADN, utilizando-se amostras de carne adquiridas num talho. Entre os protocolos testados, o método de Brometo de Cetil-Trimetil-Amónio (CTAB) modificado foi o que permitiu obter amostras de ADN com maior concentração e elevado nível de pureza. Posteriormente, foram testados e optimizados diferentes protocolos de amplificação, por PCR em tempo real, para a detecção das espécies Bos taurus (vaca), Sus scrofa (porco), Equus caballus (cavalo) e Ovis aries (ovelha). Foram empregues primers específicos de espécie para a detecção de genes mitocondriais e genómicos, consoante cada protocolo. Para o caso concreto do porco, foi efectuada a avaliação de dois protocolos, singleplex com EvaGreen® e tetraplex com AllHorse, para possível aplicação dos mesmos na sua quantificação. Os resultados demonstraram elevada especificidade e sensibilidade das reacções para esta espécie, permitindo a sua detecção até um limite de 0,001 ng e 0,1%, respectivamente. Somente a primeira metodologia se mostrou adequada para quantificação. Por último, as metodologias sugeridas foram aplicadas com sucesso na análise de 4 amostras comerciais de hambúrgueres, tendo-se verificado a consistência da rotulagem em todos os casos, no que concerne a composição em termos de espécies animais. O interesse de trabalhos neste âmbito recai na importância da autenticidade dos rótulos de produtos alimentares, principalmente nos produtos cárneos, para segurança dos consumidores e salvaguarda dos produtores.

Preferência alimentar e identificação das principais fontes de repasto sanguíneo de fêmeas Lutzomyia (Diptera: Psychodidae) em áreas endêmicas para leishmaniose visceral na grande Natal

Leishmaniasis are endemic diseases wild spread in the New and Old World, caused by the flagelated protozoan Leishmania. In the New World, the distribution of different forms of leishmaniasis is mostly in tropical regions. In the State of Rio Grande do Norte, Northeast Brazil, 85% of the captured sand flies fauna is Lutzomyia longipalpis. The distribution of the sand fly vector in the state overlaps with the disease distribution, where the presence of sand flies is associated with presence of animals shelters. The aim of this study was to analyse the blood meal preference of sand flies vector from the genus Lutzomyia spp. in laboratory conditions, to verify the vector life cicle at different temperatures sets and to identify the main blood meal source in endemic areas for visceral leishmaniasis (VL) at peri-urban regions of Natal. Sand flies samples were collected from the municipalities of São Gonçalo do Amarante and Nísia Floresta where female sand flies were grouped for the colony maintenance in the laboratory and for the analysis of the preferred source of sand fly blood meal in natural environment. The prevalence of blood meal preference and oviposition for the females sand flies was 97% for Cavia porcellus with oviposition of 19 eggs/female; 97% for Eqqus caballus with 19 eggs/female; 98% for human blood with 14 eggs/female; 71.3% for Didelphis albiventris with 8.4 eggs/female; 73% for Gallus gallus with 14 eggs/female; 86% for Canis familiaris with 10.3 eggs/female; 81.4% for Galea spixii with 26 eggs/female; 36% for Callithrix jachus with 15 eggs/female; 42.8% for Monodelphis domestica with 0% of oviposition. Female sand flies did not take a blood meal from Felis catus. Sand flies life cycle ranged from 32-40 days, with 21-50 oviposition rates approximately. This study also showed that at 32°C the life cycle had 31 days, at 28° C it had 50 days and at 22°C it increased to 79 days. Adjusting the temperature to 35°C the eggs did not hatch, thus blocking the life cycle. A total of 1540 sand flies were captured, among them, 1.310 were male and 230 were female. Whereas 86% of the sand flies captured were Lu. longipalpis as compared to 10.5% for Lu. evandroi and, 3.2% for L. lenti and 0.3% for Lu whitmani. The ratio between female and male sandfly was approximately 6 males to 1 female. In Nísia Floresta, 50.7% of the collected females took their blood meal from armadillo, 12.8% from human. Among the female sand flies captured in São Gonçalo do Amarante, 80 of them were tested for the Leishmania KDNA infectivity where 5% of them were infected with Leishmania chagasi. Female Lutzomyia spp. showed to have an opportunistic blood meal characteristic. The behavioral parameters seem to have a higher influence in the oviposition when compared to the level of total proteins detected in the host s bloodstream. A higher Lu. longipalpis life cycle viability was observed at 28°C. The increase of temperature dropped the life cycle time, which means that the life cycle is modified by temperature range, source of blood meal and humidity. Lu longipalpis was the most specie found in the inner and peridomiciliar environment. In Nísia Floresta, armadillos were the main source of blood meal for Lutzomyia spp. At São Gonçalo do Amarante, humans were the main source of blood meal due to CDC nets placed inside their houses

Patologías ováricas en equinos (Equus ferus caballus) de alto valor genético en ranchos de criadores de los departamentos de Managua, Masaya y Chinandega en el periodo Julio 2012- Julio 2013

En el presente estudio se analizó la prevalencia de patologías reproductivas y ováricas específicamente, en hembras equinas destinadas a la producción de pie de cría de alto valor genético, considerando los factores: edad, propietario, número de partos y raza sobre la presentación y desarrollo de patologías ováricas, también se realizó una evaluación del impacto financiero que representan dichos trastornos para el criador. Los datos se recopilaron de los antecedentes reproductivos y diagnósticos ultrasonográficos de distintos ranchos de los departamentos de Managua, Masaya y Chinandega, realizados en las hembras, en el periodo comprendido desde julio del 2012 hasta julio del 2013. Los datos fueron sometidos estadísticamente a análisis de varianza bajo un modelo general aditivo que resulto altamente significativo (P<0.0001) denotando que la suma de los factores considerados ejercen efecto sobre la presentación de patologías ováricas. Mediante el coeficiente de Pearson se determinó la existencia de asociación entre los factores considerados sobre la presentación de patologías ováricas y a través de la prueba de Duncan se estableció la diferencia entre medias para establecer el o los trastornos ováricos de mayor presentación. De un total de 51 ultrasonidos recopilados se encontró que del total de trastornos reproductivos (33.3%) prevalecen en mayor cantidad las patologías ováricas con un 88.23% con una prevalencia de 29.41% del total de la muestra. En tanto para las patologías ováricas analizadas se encontró que el cuerpo lúteo persistente es de mayor presentación con 26.66%, seguida por el tumor de las células de la granulosa (TCG) con un 20%, en tanto para las condiciones: ovarios multifoliculares (OMF), ovarios anéstricos (OAN) y ovarios luteinizados (OLU) fue de 13.33% en cada una; finalmente, ovarios hipoplásicos (OHP) y quistes lúteos (QUL) tuvieron el menor valor individual con un 6.66%. sobre la presentación de trastornos ováricos, ejerce alta influencia el propietario, quien responde al manejo que en general se brinda a las reproductoras en cada rancho, por otro lado se observó que las hembras jóvenes nulíparas tienden a presentar mayor prevalencia de este tipo de patologías respecto a las hembras adultas y experimentadas. El factor raza per se, no mostró inflluencia. Desde el punto de vista reproductivo, las pérdidas económicas generadas por las patologías ováricas son altas y representan un costo anual por yegua de aproximadamente $3,620.00 (C$90,717.20), en yeguas Iberoamericanas y $4,620.00 (C$115,777.20), en yeguas PRE.

Phylogeny of horse chromosome 5q in the genus Equus and centromere repositioning

Horses, asses and zebras belong to the genus Equus and are the only extant species of the family Equidae in the order Perissodactyla. In a previous work we demonstrated that a key factor in the rapid karyotypic evolution of this genus was evolutionary centromere repositioning, that is, the shift of the centromeric function to a new position without alteration of the order of markers along the chromosome. In search of previously undiscovered evolutionarily new centromeres, we traced the phylogeny of horse chromosome 5, analyzing the order of BAC markers, derived from a horse genomic library, in 7 Equus species (E. caballus, E. hemionus onager, E. kiang, E. asinus, E. grevyi, E. burchelli and E. zebra hartmannae). This analysis showed that repositioned centromeres are present in E. asinus (domestic donkey, EAS) chromosome 16 and in E. burchelli (Burchell's zebra, EBU) chromosome 17, confirming that centromere repositioning is a strikingly frequent phenomenon in this genus. The observation that the neocentromeres in EAS16 and EBU17 are in the same chromosomal position suggests that they may derive from the same event and therefore, E. asinus and E. burchelli may be more closely related than previously proposed; alternatively, 2 centromere repositioning events, involving the same chromosomal region, may have occurred independently in different lineages, pointing to the possible existence of hot spots for neocentromere formation. Our comparative analysis also showed that, while E. caballus chromosome 5 seems to represent the ancestral configuration, centric fission followed by independent fusion events gave rise to 3 different submetacentric chromosomes in other Equus lineages.

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

卡拉麦里山有蹄类自然保护区蒙古野驴(Equus hemionus)和鹅喉羚(Gazella subgutturosa)食性、种群和栖息地研究

卡拉麦里山有蹄类自然保护区是世界著名的荒漠有蹄类自然保护区, 区内有蒙古野驴(E. hemionus)、鹅喉羚(G. subgutturosa)、盘羊(Ovis ammon)以及近期野放的普氏野马(E. przewalski)。经过多年有效保护和管理，区内蒙古野驴及鹅喉羚种群得到恢复并有较大增长。从2006年开始，我们在该保护区开展了蒙古野驴和鹅喉羚的食性构成、社群结构、种群密度、卧息及采食生境利用、季节性栖息地利用等方面的研究。主要结果如下： 采用粪样显微分析技术研究了蒙古野驴、鹅喉羚与家羊、家马和家骆驼3种家养动物春季、秋季和冬季食性组成及食物生态位。结果发现：(1) 该区域的蒙古野驴、鹅喉羚和家畜所采食的植物科数和种数都不相同，针茅、驼绒藜、蒿和梭梭在各自食谱中所占的比例不同, 但蒙古野驴、鹅喉羚和家畜主要采食针茅、驼绒藜、蒿和梭梭；(2) 春季、秋季和冬季这些野生与家养草食动物两两之间的食物生态位重叠均在0.8以上，最低为0.832(冬季：鹅喉羚和家马)，最高达到0.986(秋季：蒙古野驴和家羊；秋季：家马和家羊)。五种有蹄类之间的食物生态位重叠度也达到了0.3以上。表明在卡拉麦里山有蹄类自然保护区，上述野生动物及季节性进入该自然保护区的家畜之间均存在食物竞争。冬季积雪深，食物短缺, 荒漠有蹄类易因冻饿及疾病等造成死亡。因此，应采取限制秋冬季进入该自然保护区家畜数量及调整放牧区域等保护管理措施，对该区域荒漠有蹄类实施有效保护。 对蒙古野驴和鹅喉羚的集群行为进行了初步研究。将鹅喉羚或蒙古野驴集群划分为雌性群、雄性群、母仔群、雌雄混群和独羚或独驴5种类型。共统计蒙古野驴556群次，计8235匹次。其中，雌雄混群223群次, 占40%, 为最多的集群类型；其余为雄性群(24%)、独驴(19%)、雌性群(11%)和母仔群(6% )。不同集群大小的比例差异极显著 (P=0.000)。其中2-30匹的集群占69%，独驴占18%，其余为13%。最大集群为266匹。除独驴外，其余4种集群类型的集群大小存在极显著差异。独驴作为一种特殊的集群类型，其占雄性个体的比例达63%，说明雄性个体比雌性更容易形成独驴。观察记录到鹅喉羚1286群次，计9148只次。其中，雌雄混群459群次, 占35%,为最多的集群类型；其余为独羚(18%)、母仔群(18%)、雄性群(17%)和雌性群(12%)。不同大小集群的比例差异极显著(P=0.000)，其中2一15头的集群占73%， 16头以上集群占42%，独羚仅占3%，不同集群大小差异也极为显著(P=0.000)。除独羚外，其余4种集群类型的集群大小存在极显著差异。独羚作为一种特殊的集群类型，其雌性个体的比例占到55%，因此, 不能说明雄性比雌性更容易形成独羚。 分四个季节采用截线取样法，用DISTANCE5.0估计了蒙古野驴、鹅喉羚的密度和遇见率。根据DISTANCE5.0计算，该区域春季蒙古野驴种群密度为0.55±0.14匹/km2 (平均数±标准差,下同)，夏季为0.60±0.13匹/km2，秋季为0.78±0.19匹/km2和冬季为0.54±0.14匹/km2。春季、夏季、秋季和冬季的鹅喉羚种群密度分别为1.14± 0.18头/km2，0.95±0.12头/km2，1.08±0.18头/km2和1.54±0.31头/km2。将这些结果与本区域不同时期及不同地域的蒙古野驴、鹅喉羚密度和遇见率数据进行了比较对比。截线取样法为研究干旱地区的有蹄类动物提供了一种标准方法，本研究结果为卡拉麦里自然保护区蒙古野驴和鹅喉羚的长期监测提供了基础数据。 采用样方法研究了鹅喉羚夏季和冬季卧息生境选择。夏季测定了49个卧迹样方，36个对照样方；冬季测定了75个卧迹样方，75个对照样方。研究发现，春季鹅喉羚主要选择平滩、下坡位和无坡位、海拔高度910米以上、与水源距离较远、远离道路、远离居民点、隐蔽级高、中低植被密度和中高草本密度的区域作为卧息生境；而冬季则主要选择山坡、阳坡和半阴半阳坡、中上坡位和下坡位、海拔900～1000m范围、离道路501～1000m以及大于2000m的距离、靠近居民点、中低隐蔽级、中厚度(1.1-3cm)雪深、中高植被密度和中高草本密度的区域作为卧息生境。主成分分析表明，卡拉麦里山鹅喉羚夏季季卧息样方前4个主成分的累积贡献率达到86.57％，第1主成分主要反映卧迹样方的植物密度、草本密度、针茅密度、至最近居民点距离、至永久水源最近距离和海拔。冬季该区域鹅喉羚卧息样方前4个主成分的累积贡献率达到73.88％，第1主成分主要反映卧迹样方的植物密度、草本密度、针茅密度和坡度。 通过设立样方研究了蒙古野驴的采食生境。研究发现，夏季和秋季两个季节蒙古野驴主要在平滩和山沟采食。集中在无坡向的平滩、1001m以上的海拔高度、与水源距离较近、远离道路、远离矿区等人类活动点、植被盖度高、草本盖度高、植物多样性高、草本多样性高(P＜0.05)的区域采食。主成分分析表明，夏季影响蒙古野驴采食生境选择的环境因子是植物盖度、草本盖度、针茅盖度、植物密度、草本密度、针茅密度、植物种类、草本种类和禾本科种类。秋季影响蒙古野驴采食生境选择的环境因子是海拔、植物盖度、草本盖度、针茅盖度、植物密度、草本密度、针茅密度、植物种类、草本种类和禾本科种类。 利用卡山保护区不同季节野外种群调查数据，以3S技术为手段，评价了蒙古野驴和鹅喉羚季节性栖息地适宜性。首先把各季节蒙古野驴和鹅喉羚分布点图层，分别和固定水源点图层、道路图层、固定冬牧点图层和矿点图层进行叠加和距离查询，用Vanderploeg & Scavia’s选择指数(Ei*)分析了蒙古野驴和鹅喉羚季节性栖息地选择及其主要影响因素，建立了固定水源点、植被、道路、固定冬牧点和矿点对蒙古野驴和鹅喉羚栖息地影响强度的评价标准。通过缓冲区分析，分别研究了固定水源点、道路、固定冬牧点、矿点对蒙古野驴和鹅喉羚季节性栖息地的影响。通过缓冲区分析，综合研究了与人类活动有关的道路、固定冬牧点和矿点因子对蒙古野驴和鹅喉羚季节性栖息地的影响。最后将固定水源点、植被、道路、固定冬牧点和矿点等5个因子结合起来通过地图综合查询研究分析了这些因子对蒙古野驴和鹅喉羚季节性栖息地的综合影响，在固定水源点较适宜以上范围给出了该保护区蒙古野驴和鹅喉羚季节性不同适宜等级的生境面积。 我们建议采取以下积极措施来保护卡山保护区荒漠生态系统多样性以有效保护该区域内栖息的蒙古野驴和鹅喉羚：提高保护区周边社区政府的认识，提高公众参与自然资源和生物多样性保护的意识；加强水源地建设，积极采取飞播等措施,恢复植被，提高栖息生境质量；逐步限制进入卡拉麦里山越冬渡春的家畜数量；严格进入卡山保护区采矿制度，取缔非法矿业；加强草原围栏生态通道建设；与蒙古国合作建立跨国界的国际自然保护区；建立卡山自然保护区自然资源有偿使用机制；开展长期蒙古野驴和鹅喉羚种群监测。

L'equus eroticus ou l'image de la femme qui chevauche l'homme dans la gravure européenne au XVIe siècle : érotisme ou propagande antiféministe ?

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infertilidade associada a defeito microtubular dos espermatozóides de jumento (Equus asinus) avaliados por microscopia eletrônica de transmissão

Descreve-se um caso de infertilidade de um jumento SRD confirmada por meio de microscopia eletrônica de transmissão (MET). O espermiograma, avaliado sob microscopia ótica, revelou baixa motilidade e alta concentração de anormalidades espermáticas do tipo gota citoplasmática proximal. O material foi avaliado por MET, observando-se um acúmulo desordenado de microtúbulos causando protusões irregulares na região do colo espermático. O último teste realizado correspondeu ao de fertilidade in vivo, utilizando-se quatro éguas portadoras de bom histórico reprodutivo, nas quais não foi possível confirmar nenhuma prenhez. Frente aos resultados obtidos, associados aos achados da MET, estabeleceu-se o diagnóstico de infertilidade associada a defeito microtubular dos espermatozóides.

Avaliaçãeo ultrassonográfica da dinâmica folicular ovariana de jumenta (equus asinus) durante o ciclo estral

The ovarian activity was accessed by ultrasound throughout 10 estrous cycles from Jennies (Marchador Brasileira). Daily ultrasound examinations were performed and the dada was retrospectively studied based on daily identification of each follicle detected. Blood samples were collected every 24 hours from ovulation (D0) until the next identified ovulation. The follicles measuring 11mm were detected and their mean diameter was registered daily using an ovarian map and permitting a retrospective evaluation of the dada, which represented the follicular growth dynamics. One and two major follicular waves were detected in six (60%) and four (40%) cycles respectively from ten estrous cycles in the present study. The primary wave emergency and follicle deviation from the cycles observed during the present study with one major follicular wave occurred at day 10.2 ± 0.75 and at day14.1 ± 0.81 and for cycles with two major waves, those events occurred at 9.0 ± 2.0 and 14.0 ± 1.15 days after ovulation. The maximum diameter of the dominant follicle at the cycles with one and two follicular waves were 37.2 ± 3.35 mm and 37.3 ± 1.1, respectively. The mean intervals from two ovulations were 23.0 ± 1.79 and 22.3 ± 1.26 days when observed in cycles with one and two major follicular waves. The maximum diameter of the dominant follicle was slightly smaller when double ovulations were observed if compared with the single ovulations (P > 0.05). It can be concluded that wave emergency period is observed around 9.6 days of the estrous cycle and the occurrence of deviation can be observed on day 14 of the estrous cycle.

Concentraçãeo plasmática de progesterona durante o ciclo estral de jumentas (equus asinus) com ovulaçõees simples e duplas

Ten estrous cycles of seven Machador Brasileiro jennies located at Estaçãeo Experimental de Zootecnia - Colina - Sãeo Paulo were monitored and the plasmatic progesterone concentration determined by the radioimmunoassay method in solid phase, employing commercial kits (DPC). For progesterone concentration determination and CL diameter there were excluded the animals which presented diestrus ovulations. The corpora lutea formed from single ovulations or double ovulations presented a mean diameter of 26.2 ± 4.4 and 22.1 ± 2.7 mm, respectively, which represented 66.1% and 64.1% of the preovulatory follicle diameter. The presence of two CLs from double ovulations was not reflected by a significant increase on progesterone concentrations.