Philosophiae Doctor - PhD / The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies. Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P ≤ 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum. It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies

1 AbstractAbstractAbstractAbstract Proteins secreted in the male reproductive tract play a key role in post-testicular development of sperm and in further steps needed for fertilization. Sperm maturation represents a key step in the reproduction process. Sperm, during the passage through the epididymis undergoes significant changes due to proteolytic and glycolytic activities in the epididymal fluid. Inhibitor of acrosin protects spermatozoa and reproductive epithelium against proteolytic degradation and also protects binding sites for ZP on sperm plasma membrane. In boar reproductive system acrosin inhibitor (AI) was found in seminal plasma and on sperm plasma membrane. Polyclonal antibody recognized AI in extracts of the cauda epididymidis, seminal vesicles, prostate, and Cowper's glands. Using immunofluorescence method has revealed the AI in the epithelium and lumen of these organs but also on the surface of epididymal and ejaculated spermatozoa. We registered the increasing signal of AI from caput to cauda epididymis. Gene expression of AI mRNA was detected in the epididymis, seminal vesicles, prostate, and Cowper's glands and increased gradually throughout the epididymal duct. In present study, we also monitored AI in boar epididymal fluid and spermatozoa along the organ. In the epididymis, AI may...

The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway. / Ph. D.

Master of Science / Department of Anatomy and Physiology / Bruce D. Schultz / Epithelial cells lining the vas deferens modify the luminal contents to which sperm are exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and identity of vas deferens epithelial potassium channels that provide the correct luminal environment for sperm maturation and delivery have not yet been determined. Cultures of vas deferens epithelial cells isolated from adult pigs were employed to investigate contributions of selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine, a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin- stimulated short circuit current, Isc, in a concentration dependent manner with a maximum inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine, lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a concentration dependent manner. Fitting a modified Michalis-Menten function to the data revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2 immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may account for the reportedly high concentration of potassium in the male reproductive duct lumen. TASK-2 likely contributes to male fertility as an integral member of the regulated transport processes that account for the luminal environment to which sperm are exposed.

Objetivo: este estudo teve como objetivo avaliar as infecções por papilomavírus humano (HPV) e Chlamydia trachomatis (CT) em pacientes com artrite reumatoide (AR) pré e pós-terapia anti-TNF. Método: foram avaliadas 50 pacientes do sexo feminino com AR (preenchiam os critérios do Colégio Americano de Reumatologia) que eram elegíveis para terapia anti-TNF. Cinquenta pacientes foram incluídas no estudo de forma prospectiva. Destas, 45 pacientes foram reavaliadas após 6 meses de terapia anti-TNF. Inicialmente as 50 pacientes com AR foram comparadas com 50 controles saudáveis pareadas por idade. Foram avaliados dados demográficos, avaliação ginecológica (citologia cervical e avaliações histológicas), função sexual, parâmetros de doença e tratamento atual da AR. Os testes para detecções dos DNAs do HPV e CT nas espécimes cervicais foram realizados utilizando a captura híbrida II. Resultados: na avaliação inicial, a mediana da idade das pacientes com AR e controles foi de 49 (18-74) vs. 49 (18-74) anos, p = 1,0. Observou-se uma tendência de menor frequência de infecção por HPV nas pacientes com AR pré anti-TNF em relação aos controles (14% vs. 30%, p = 0,054). Adicionalmente, realizou-se avaliação das pacientes com AR com infecção positiva e negativa por HPV antes da terapia anti-TNF que demonstrou que o primeiro grupo apresentou maior frequência de relações sexuais (100% vs. 48%, p = 0,014), maior número de parceiros sexuais [1 (1-1) vs. 0 (0-1), p = 0,032] e maior frequência de citologia cervical anormal (43% vs. 7%, p = 0,029). A idade atual, a duração da doença, os parâmetros da doença e os tratamentos foram semelhantes em ambos os grupos (p > 0,05). Após 6 meses de terapia anti-TNF, a infecção por HPV permaneceu inalterada em cinco pacientes, enquanto que dois tornaram-se negativos e uma paciente adicional tornou-se positiva (p = 1,0). A infecção por CT foi uniformemente negativa nas pacientes com AR pré e pós-TNF, assim como nas controles. Conclusões: a infecção por HPV observada nas pacientes sexualmente ativas com AR antes da terapia anti-TNF foi leve, sem evidência de infecção por CT. A terapia anti-TNF não aumentou o risco de exacerbação e/ou progressão das infecções por HPV e CT em pacientes com AR / Objective: to evaluate human papillomavirus (HPV) and Chlamydia trachomatis (CT) infections in rheumatoid arthritis (RA) patients pre- and post-TNF blocker. Methods: fifty female RA patients (American College of Rheumatology criteria), who were eligible to anti-TNF therapy, [n = 50 at baseline (BL) and n = 45 after 6 months of treatment (6M)] and 50 agematched healthy controls were prospectively enrolled. They were assessed for demographic data, gynecologic, sexual, cervical cytology and histological evaluations, disease parameters and current treatment. HPV DNA and CT DNA testing in cervical specimens were done using Hybrid Capture II assays. Results: at BL, the median current age of RA patients and controls was 49(18-74) vs. 49(18-74) years, p = 1.0. A trend of lower frequency of HPV infection was observed in AR patients pre anti-TNF compared to controls (14% vs. 30%, p = 0.054). Further evaluation of AR patients with and without HPV infection before anti-TNF therapy showed that the former group had higher frequency of sexual intercourses (100% vs. 48%, p = 0.014), higher median number of sexual partners [1(1-1) vs. 0(0-1), p=0.032] and higher frequency of abnormal cervical cytology (43% vs. 7%, p = 0.029). Current age, disease duration, disease parameters and treatments were alike in both groups (p > 0.05). At 6M after TNF blockage, HPV infection remained unchanged in five patients, whereas two became negative and one additional patient turn out to be positive (p = 1.0). CT infection was uniformly negative in RA patients pre- and post-TNF blockage and in controls. Conclusions: anti-TNF does not seem to increase short-term risk of exacerbation and/or progression of HPV and CT infections in RA patients

Além da deficiência física, a diminuição ou perda da sensibilidade geniturinária é um dos maiores impactos para mulheres com Lesão Medular (LM). Devido à perda da mobilidade funcional e as barreiras arquitetônicas, estas muitas vezes não tem acesso aos cuidados adequados para a saúde ginecológica. Como aproximadamente 80% das lesões da medula espinal acometem indivíduos do sexo masculino, os estudos raramente focam as necessidades e questões referentes às mulheres. Objetivo: Avaliar a prevalência de infecções genitais não virais em mulheres com deficiência física por lesão medular, comparativamente às mulheres saudáveis Método: Estudo de corte transversal, caso controle. Foram estudadas 52 mulheres com LM (grupo estudo) e 57 mulheres saudáveis (grupo controle). Todas responderam a um questionário estruturado e foram submetidas à coleta de conteúdo vaginal para pesquisa de Trichomonas vaginalis e leveduras, bacterioscopia com coloração pelo método Gram, cultura geral (meio ágar sangue), cultura para fungos (meio Sabouraud) e coleta de conteúdo endocervical para pesquisa de Chlamydia trachomatis e Neisseria gonorrhorae (reação em cadeia da polimerase) e cultura para Mycoplasmas sp (meios U9, A7). Resultados: As mulheres com lesão medular, comparativamente ao grupo controle, apresentaram maior frequência de Candida sp no exame micológico direto (p= 0,017); entretanto não foi observada diferença estatisticamente significativa na frequência de isolamento de espécies fúngicas entre os grupos. O grupo estudo apresentou maior frequência de isolamento de Escherichia coli (p= 0,002) e de Corynebacterium sp (p= 0,023) e menor frequência de Lactobacillus sp (p < 0,001) em conteúdo vaginal. Em ambos os grupos não foram encontrados casos positivos para Trichomonas vaginalis. A avaliação do escore de Nugent para diagnóstico de vaginose bacteriana demonstrou maior freqüência de flora intermediária (Nugent 4-7) no grupo estudo (p= 0,039). As pesquisas de Chlamydia trachomatis e Neisseria gonorroheae foram negativas em todas as mulheres. Com relação ao isolamento de Mycoplasmas sp, os resultados foram semelhantes em ambos os grupos. Conclusão: A menor freqüência de isolamento de Lactobacillus sp e a maior freqüência de isolamento de Corynebacterium sp e de Escherichia coli na vagina em mulheres com LM, assim como a elevada frequência de flora intermediária pelo escore de Nugent verificada nas mesmas, fortemente sugerem um desequilíbrio da microbiota vaginal, diferente de uma flora dominada por Lactobacillus sp em tais mulheres. Desde que os Lactobacillus sp são essenciais para a manutenção da flora vaginal e a inibição do crescimento de outras bactérias, sua ausência relativa em mulheres com LM pode influenciar a ocorrência de infecções do trato urogenital. Adicionalmente, a mais elevada frequência de detecção de fungos pela microscopia em mulheres com LM sugere que estas podem albergar uma maior concentração vaginal desses microorganismos do que outras mulheres / Besides their physical disability, decreased or absent genitourinary sensitivity has a huge impact in women with spinal cord injury (SCI). Due to the absence of functional mobility and the architectonic barriers these women frequently do not have access to adequate gynecological care. Since about 80% of spinal cord injuries affect men, studies have rarely focused on the needs of women with SCI. Objective: To evaluate the prevalence of non-viral genital infections in women with SCI compared to mobile women. Methods: Fifty two women with SCI (study group) and 57 mobile women (control group) were evaluated in a case-control study. All answered a structured questionnaire and were submitted to the following microbiological tests: fresh examination of vaginal secretions for Trichomonas vaginalis and yeasts, Gram stain, general culture (agar-blood medium), yeast culture (Sabouraud medium) and endocervical sampling for Chlamydia trachomatis and Neisseria gonorrhorae (polymerase chain reaction) and Mycoplasmas sp. (U9, A7 medium). Results: A higher percentage of women with SCI had Candida sp detected by direct mycological examination than did women in the control group (p= 0.017). However there were no significant differences between the two groups in the frequency of yeast-positive cultures. The study group had a higher isolation frequency from the vagina of Escherichia coli (p= 0.002) and Corynebacterium sp (p= 0.023) and a lower frequency of Lactobacillus sp (p < 0.001). In both groups, there were no cases positive for T. vaginalis, C. trachomatis or N. gonorrhoeae. The evaluation of Nugent score for bacterial vaginosis showed a higher frequency of intermediate flora (Nugent score 4-7) in the study group (p= 0.039). Related to Mycoplasma sp isolation, the results were similar in both groups. Conclusion: The lower frequency of Lactobacillus sp isolation and the higher frequency of Corynebacterium sp and Escherichia coli isolation from the vagina in women with SCI, and the higher frequence of intermediate Nugent score, strongly suggests a disequilibrium of the vaginal microbiota away from a Lactobacillus sp dominated flora in these women. Since lactobacilli are essential for maintaining vaginal health and inhibiting growth of other bacteria, their relative absence in women with SCI may influence the occurrence of urogenital tract infections in these women. The higher frequency of yeast detection by microscopy in women with SCI suggests that these women may harbor a higher vaginal yeast concentration than do other women

Infertility affects 15 % of couples, which corresponds to 60 - 80 million worldwide. The microenvironments in which the oocyte, embryo and fetus mature are vital to the establishment and development of a healthy pregnancy. Different biological systems, such as angiogenesis, the immune system and apoptosis need to be adequately regulated for pregnancy to occur and progress normally. The overall aim of this thesis was to investigate the impact of Histidine-rich glycoprotein (HRG) and Src homology 2 domain-containing adapter protein B (SHB) on human female fertility. HRG is a plasma protein that regulates angiogenesis, the immune system, coagulation/fibrinolysis and apoptosis, by building complexes with various ligands. The impact of HRG on fertility is studied here for the first time. HRG is present in follicular fluid, the Fallopian tube, endometrium, myometrium and placenta. HRG distribution within embryo nuclei depends on developmental stage. Blastocysts express and secrete HRG. The HRG C633T single nucleotide polymorphism (SNP) appears to affect the chance of pregnancy and, correspondingly, parameters associated with pregnancy in IVF. Additionally, this HRG genotype may increase the risk in IVF of only developing embryos unfit for transfer. SHB is an adaptor protein involved in intracellular signaling complexes that regulate angiogenesis, the immune system and cell proliferation/apoptosis. Shb knockout mice have altered oocyte/follicle maturation and impaired embryogenesis. The impact of three SHB polymorphisms (rs2025439, rs13298451 and rs7873102) on human fertility is studied for the first time. The SNP prevalences did not differ between infertile and fertile women. BMI, gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer seems to be affected by SHB genotype. In conclusion, HRG and SHB appear to influence female fertility. They are potential biomarkers that might be used for predicting pregnancy chance in infertile women. Knowledge of these genotypes may improve patient counseling and individualization of treatment.

Com o objetivo de acasalar novilhas aos 14/15 meses de idade, cem bezerras Hereford foram submetidas a dois sistemas de alimentação (SA) de 20/06 a 20/08: a) PN=50 bezerras em pastagem natural; b) PNs=50 bezerras em pastagem natural suplementadas (14% PB; 72% NDT) a 1%/dia do peso corporal (PC). Em 20/08/2012, em lote único, pastejaram azevém (PA). Quinze dias antes do final deste periodo (18/10), metade das novilhas de cada SA passaram a receber a suplementação do PNs, estendendo-se por quinze dias no retorno à PN (03/11), constituindo novos SA: c) PNs-PAs: suplementadas em PN e por 15 dias em PA + 15 dias em PN; d) PNs-PA: suplementadas em PN; e) PN-PAs: suplementadas por 15 dias em PA + 15 dias em PN; f) PN-PA: sem suplementação. O período reprodutivo (PR) foi em PN. O escore de trato reprodutivo (ETR-US) e a área pélvica (APR) foram determinados aos 365 dias de idade. De 20/06 a 20/08, o ganho diário médio (GDM) e o peso corporal (PC) das bezerras do PNs foram 67,71% e 9,95% e 6,19% e 15,46% superiores, respectivamente, às do PN. O PNs também apresentou área do músculo longissimus (AOLUS) superior em 8,48%, 22,91% e 21,82%, respectivamente, ao PN. A suplementação proporcionou maior espessura de gordura subcutânea (EGSUS), 95,12% superior em relação ao PN. Em pastagem de azevém, na primeira pesagem, o PNs apresentou 5% de GDM superior às do PN. Bezerras do PNs apresentaram PC 13,36% superior ao PN e 18,20% e 13,33% de AOLUS superiores, respectivamente, aos valores do PN. O PNs obteve maior EGSUS, 1,43±0,67 e 0,89±0,75 mm, superando as do PN. Com o objetivo de acasalar novilhas aos 14/15 meses de idade, cem bezerras Hereford foram submetidas a dois sistemas de alimentação (SA) de 20/06 a 20/08: a) PN=50 bezerras em pastagem natural; b) PNs=50 bezerras em pastagem natural suplementadas (14% PB; 72% NDT) a 1%/dia do peso corporal (PC). Em 20/08/2012, em lote único, pastejaram azevém (PA). Quinze dias antes do final deste periodo (18/10), metade das novilhas de cada SA passaram a receber a suplementação do PNs, estendendo-se por quinze dias no retorno à PN (03/11), constituindo novos SA: c) PNs-PAs: suplementadas em PN e por 15 dias em PA + 15 dias em PN; d) PNs-PA: suplementadas em PN; e) PN-PAs: suplementadas por 15 dias em PA + 15 dias em PN; f) PN-PA: sem suplementação. O período reprodutivo (PR) foi em PN. O escore de trato reprodutivo (ETR-US) e a área pélvica (APR) foram determinados aos 365 dias de idade. De 20/06 a 20/08, o ganho diário médio (GDM) e o peso corporal (PC) das bezerras do PNs foram 67,71% e 9,95% e 6,19% e 15,46% superiores, respectivamente, às do PN. O PNs também apresentou área do músculo longissimus (AOLUS) superior em 8,48%, 22,91% e 21,82%, respectivamente, ao PN. A suplementação proporcionou maior espessura de gordura subcutânea (EGSUS), 95,12% superior em relação ao PN. Em pastagem de azevém, na primeira pesagem, o PNs apresentou 5% de GDM superior às do PN. Bezerras do PNs apresentaram PC 13,36% superior ao PN e 18,20% e 13,33% de AOLUS superiores, respectivamente, aos valores do PN. O PNs obteve maior EGSUS, 1,43±0,67 e 0,89±0,75 mm, superando as do PN. / Aiming at breeding heifers with 14-15 months of age, 100 Hereford calves were submitted to two feeding systems (FS) between 06/20 and 08/20: a) NP=50 calves grazing on natural pasture and b) NPs=50 calves grazing on natural pasture and fed a supplement (14% CP; 72% TDN) at 1% body weight (BW)/day. On 08/20, heifers were transferred as single group to a ryegrass (RG) pasture. During the last 15 days of ryegrass grazing (10/18) and the first 15 days upon returning to NP (11/03), half of the heifers in each previous FS were fed the supplement, thereby establishing new FS: c) NPs-RGs: previously supplemented on NP and supplemented for 15 days on RG + 15 days on NP; NPs-RG: previously supplemented on NP; e) NP-RGs: supplemented only for 15 days on RG + 15 days on NP; and f) NP-RG: not supplemented in either period. During the breeding season (BS), heifers grazed on NP. Reproductive tract score (TRS) and relative pelvic area (RPA) were determined at 365 days of age. On 07/20 and 08/20, NPs heifers presented 67.71% and 9.95% and 6.19% and 15.46% higher daily weight gain (DWG) and BW than NP heifers, as well as 22.91% and 21.82% larger loin eye area (UREA) and 95.12% average thicker backfat (UFT). At the end of the grazing period, NPs heifers were, on average, 13.36% heavier, and presented higher average UFT than NP (1.43±0.67 vs. 0.89±0.75). Between 10/18 and 11/18, NPs-RGs and NP-RGs heifers presented higher DWG and the highest UFT values were obtained in NPs-RGs heifers. At the start of the BS, NPs-RGs had higher BCS and BW. RPA of NPs-RGs (106.41±11.92 cm²) was only different from NP-RGs (93.74±11.76 cm²). The highest percentage of puberty was determined in NPs-RG (34.3%). The presence of CL was detected in 17.8% of the heifers. Pregnancy rates of 47%, 22%, 18%, and 13% were obtained in NPs-RGs, NPs-RG, NP-RGs, and NP-RG heifers, respectively. Pregnant heifers presented higher DWG between 10/18 and 11/18, and higher RPA, BW, and BCS at the start and end of the BS. Higher pasture DM content and higher supplement levels than those evaluated in the present study are required to achieve better calf development and better reproduction indices when aiming at breeding heifers at 14-15 months of age.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of the present study was to evaluate the reflection of supplementation with integral rice bran and/or protected fat in the productive parameters of oat and ryegrass pasture and in the development, reproductive performance and behavior of beef heifers. Twenty-eight heifers, Charolais x Nellore crossbred, with initial average age of 18 moths and initial average weight of 274.9 kg, were distributed into four treatments with three repetitions per area. The animals were kept in oat + ryegrass pasture and distributed into the following treatments: Without supplementation (SS): heifers kept in pasture; Megalac (MEG): supplementation with protected fat; Integral rice bran (FAI): supplementation with integral rice bran; FAI+MEG: supplementation with integral rice bran + protected fat. The pasture intake increased throughout the periods (Y=2.71542+.01665*Day; P=.0023). The animal stocking, stocking rate, loss of forage and pasture intake weren´t influenced by treatments (P>.05). Body condition of heifers increased linearly .012 points every day, correlating positively with the final weight (r=.79; P<.0001). The greatest daily weight gain, 1.395 kg, occurred at the first period and when animals were supplemented with FAI+MEG. At the last period, animals from SS treatment had the lowest daily weight gain, .888 kg. Neutral detergent fiber intake increased linearly .045 kg per day (P<.05) during grazing periods. The supplementation with FAI and FAI+MEG reduced the time spent by heifers with grazing, 49.63%, in relation to animals from SS and MEG treatments, 63.13%. The feeding stations per minute had an increase during grazing periods due to the reduction in time spent in each feeding station and the linear decrease of bit number per feeding station (P<.05), the latter ranging 34.48% lower at the final of grazing period. The supplementation of beef heifers with integral rice bran and/or protected fat doesn‟t alter productive parameters of oat and ryegrass pasture, neither pasture intake and total dry matter intake. Increases in daily accumulation of dry matter and feeding conversion promoted an increase in forage losses. The average daily weight gain is not changed by the supplementation. The use of pasture of temperate climate, supplemented or not, promotes adequate structural and reproductive development of the heifers, given benefiting the zootecnic indices at the first mating at 25/27 months of age. The heifers supplemented with FAI and FAI+MEG require less time for activities of grazing seizure. Grazing and idle times don‟t change with periods of grazing, however rumination time grows with the increase of neutral detergent fiber intake. / O objetivo do presente estudo foi avaliar o reflexo da suplementação com farelo de arroz integral e/ou gordura protegida nos parâmetro produtivos da pastagem de aveia e azevém e no desenvolvimento, desempenho reprodutivo e comportamento de novilhas de corte. Utilizaram-se 28 novilhas cruzas Charolês x Nelore com idade média inicial de 18 meses e peso vivo inicial médio de 274,9 kg, divididas em quatro tratamentos com três repetições por área. Os animais foram mantidos em pastagem de aveia + azevém e distribuídos nos seguintes tratamentos: Sem suplementação (SS): novilhas mantidas exclusivamente em pastagem; Megalac (MEG): suplementação com gordura protegida; Farelo de arroz integral (FAI): suplementação com FAI; FAI+MEG: suplementação FAI mais gordura protegida. O consumo do pasto foi crescente ao longo dos períodos (Y=2,71542+0,01665*Dia; P=0,0023). A carga animal, lotação, perdas de forragem e consumo do pasto não sofreram efeito dos tratamentos (P>0,05). A condição corporal das novilhas aumentou linearmente, com acréscimo a cada dia de 0,012 pontos, correlacionando-se positivamente com o peso final (r=0,79; P<0,0001). O maior ganho de peso diário, 1,395 kg, ocorreu no primeiro período quando os animais foram suplementados com FAI+MEG. No último período os animais SS apresentaram o menor ganho de peso diário, 0,888 kg. O consumo de fibra detergente neutro aumentou linearmente (P<0,05) em 0,045 kg por dia no decorrer dos períodos de pastejo. A suplementação com FAI e FAI+MEG reduziu o tempo despendido pelas novilhas para pastejo, 49,63%, em relação aos animais SS e MEG, 63,13%. As estações alimentares por minuto tiveram aumento crescente no decorrer do período de pastejo com redução no tempo gasto em cada estação alimentar e decréscimo linear no número de bocados por estação alimentar (P<0,05), sendo a variação deste último de 34,48% a menos no final do período de pastejo. A suplementação de novilhas de corte com FAI e/ou gordura protegida não altera os parâmetros produtivos da pastagem de aveia e azevém, nem o consumo do pasto e o consumo total de matéria seca. Aumentos da taxa de acúmulo diário de matéria seca e da carga animal promovem aumento das perdas de forragem. O ganho de peso diário não é alterado por ocasião da suplementação. O uso de pastagem temperada suplementada ou não, promove adequado desenvolvimento estrutural e do trato reprodutivo das novilhas, beneficiando os índices zootécnicos no primeiro acasalamento aos 25/27 meses de idade. Novilhas suplementadas com FAI e FAI+MEG demandam menos tempo para a atividade de pastejo e aumentam seu período de ócio, sem modificar seus padrões de deslocamento dentro do piquete e apreensão do pasto. O tempo de pastejo e ócio não sofrem alterações nos distintos períodos de utilização da pastagem, porém, o tempo de ruminação aumenta com o aumento da ingestão da fibra detergente neutro.

Com o objetivo de acasalar novilhas aos 14/15 meses de idade, cem bezerras Hereford foram submetidas a dois sistemas de alimentação (SA) de 20/06 a 20/08: a) PN=50 bezerras em pastagem natural; b) PNs=50 bezerras em pastagem natural suplementadas (14% PB; 72% NDT) a 1%/dia do peso corporal (PC). Em 20/08/2012, em lote único, pastejaram azevém (PA). Quinze dias antes do final deste periodo (18/10), metade das novilhas de cada SA passaram a receber a suplementação do PNs, estendendo-se por quinze dias no retorno à PN (03/11), constituindo novos SA: c) PNs-PAs: suplementadas em PN e por 15 dias em PA + 15 dias em PN; d) PNs-PA: suplementadas em PN; e) PN-PAs: suplementadas por 15 dias em PA + 15 dias em PN; f) PN-PA: sem suplementação. O período reprodutivo (PR) foi em PN. O escore de trato reprodutivo (ETR-US) e a área pélvica (APR) foram determinados aos 365 dias de idade. De 20/06 a 20/08, o ganho diário médio (GDM) e o peso corporal (PC) das bezerras do PNs foram 67,71% e 9,95% e 6,19% e 15,46% superiores, respectivamente, às do PN. O PNs também apresentou área do músculo longissimus (AOLUS) superior em 8,48%, 22,91% e 21,82%, respectivamente, ao PN. A suplementação proporcionou maior espessura de gordura subcutânea (EGSUS), 95,12% superior em relação ao PN. Em pastagem de azevém, na primeira pesagem, o PNs apresentou 5% de GDM superior às do PN. Bezerras do PNs apresentaram PC 13,36% superior ao PN e 18,20% e 13,33% de AOLUS superiores, respectivamente, aos valores do PN. O PNs obteve maior EGSUS, 1,43±0,67 e 0,89±0,75 mm, superando as do PN. Com o objetivo de acasalar novilhas aos 14/15 meses de idade, cem bezerras Hereford foram submetidas a dois sistemas de alimentação (SA) de 20/06 a 20/08: a) PN=50 bezerras em pastagem natural; b) PNs=50 bezerras em pastagem natural suplementadas (14% PB; 72% NDT) a 1%/dia do peso corporal (PC). Em 20/08/2012, em lote único, pastejaram azevém (PA). Quinze dias antes do final deste periodo (18/10), metade das novilhas de cada SA passaram a receber a suplementação do PNs, estendendo-se por quinze dias no retorno à PN (03/11), constituindo novos SA: c) PNs-PAs: suplementadas em PN e por 15 dias em PA + 15 dias em PN; d) PNs-PA: suplementadas em PN; e) PN-PAs: suplementadas por 15 dias em PA + 15 dias em PN; f) PN-PA: sem suplementação. O período reprodutivo (PR) foi em PN. O escore de trato reprodutivo (ETR-US) e a área pélvica (APR) foram determinados aos 365 dias de idade. De 20/06 a 20/08, o ganho diário médio (GDM) e o peso corporal (PC) das bezerras do PNs foram 67,71% e 9,95% e 6,19% e 15,46% superiores, respectivamente, às do PN. O PNs também apresentou área do músculo longissimus (AOLUS) superior em 8,48%, 22,91% e 21,82%, respectivamente, ao PN. A suplementação proporcionou maior espessura de gordura subcutânea (EGSUS), 95,12% superior em relação ao PN. Em pastagem de azevém, na primeira pesagem, o PNs apresentou 5% de GDM superior às do PN. Bezerras do PNs apresentaram PC 13,36% superior ao PN e 18,20% e 13,33% de AOLUS superiores, respectivamente, aos valores do PN. O PNs obteve maior EGSUS, 1,43±0,67 e 0,89±0,75 mm, superando as do PN. / Aiming at breeding heifers with 14-15 months of age, 100 Hereford calves were submitted to two feeding systems (FS) between 06/20 and 08/20: a) NP=50 calves grazing on natural pasture and b) NPs=50 calves grazing on natural pasture and fed a supplement (14% CP; 72% TDN) at 1% body weight (BW)/day. On 08/20, heifers were transferred as single group to a ryegrass (RG) pasture. During the last 15 days of ryegrass grazing (10/18) and the first 15 days upon returning to NP (11/03), half of the heifers in each previous FS were fed the supplement, thereby establishing new FS: c) NPs-RGs: previously supplemented on NP and supplemented for 15 days on RG + 15 days on NP; NPs-RG: previously supplemented on NP; e) NP-RGs: supplemented only for 15 days on RG + 15 days on NP; and f) NP-RG: not supplemented in either period. During the breeding season (BS), heifers grazed on NP. Reproductive tract score (TRS) and relative pelvic area (RPA) were determined at 365 days of age. On 07/20 and 08/20, NPs heifers presented 67.71% and 9.95% and 6.19% and 15.46% higher daily weight gain (DWG) and BW than NP heifers, as well as 22.91% and 21.82% larger loin eye area (UREA) and 95.12% average thicker backfat (UFT). At the end of the grazing period, NPs heifers were, on average, 13.36% heavier, and presented higher average UFT than NP (1.43±0.67 vs. 0.89±0.75). Between 10/18 and 11/18, NPs-RGs and NP-RGs heifers presented higher DWG and the highest UFT values were obtained in NPs-RGs heifers. At the start of the BS, NPs-RGs had higher BCS and BW. RPA of NPs-RGs (106.41±11.92 cm²) was only different from NP-RGs (93.74±11.76 cm²). The highest percentage of puberty was determined in NPs-RG (34.3%). The presence of CL was detected in 17.8% of the heifers. Pregnancy rates of 47%, 22%, 18%, and 13% were obtained in NPs-RGs, NPs-RG, NP-RGs, and NP-RG heifers, respectively. Pregnant heifers presented higher DWG between 10/18 and 11/18, and higher RPA, BW, and BCS at the start and end of the BS. Higher pasture DM content and higher supplement levels than those evaluated in the present study are required to achieve better calf development and better reproduction indices when aiming at breeding heifers at 14-15 months of age.