Dissertationen zum Thema „Ovine pregnancy“

Ovine placental lactogen (oPL) is postulated to be involved in the repartitioning of maternal nutrients during pregnancy, through its effect on insulin metabolism. Ovine pancreatic insulin responses to exogenous glucose are depressed during pregnancy and this depression becomes more pronounced as gestation advances. In addition, under the hormonal environment of rising oPL and growth hormone (oGH) concentrations, maternal whole body glucose irreversible loss (GIL) increases. The percentage of GIL accounted for by uterine glucose uptake also increases with advancing gestation and increasing litter size. Regression analysis of oPL concentration with glucose uterine uptake as a percentage of GIL, accounts for 39% of variation. Maternal oPL concentrations which increase with gestational age, were significantly greater in multiple bearing ewes and ewes subjected to reduced metabolisable energy (ME) intakes. It is postulated that through actions on pancreatic sensitivity, oPL plays a major role as a homeorhetic control during pregnancy. Elevated oPL concentrations were strongly associated with continually depressed pancreatic insulin secretory ability. The reduction in pancreatic sensitivity to glucose was not as a result of elevation in GH or non-esterified fatty acid (NEFA) concentrations. Muscle insulin receptor number and affinity were found to increase with increasing litter size, suggesting that pregnancy associated insulin resistance occurs predominantly in adipose tissue. During ovine pregnancy there is a specific stimulation of maternal gluconeogenesis. As gestation advances, an increasingly greater proportion of this glucose is partitioned to the gravid uterus. The development of insulin resistance, together with the suppression of pancreatic activity, ensures the preferential uptake of glucose by non-insulin dependent tissues over insulin dependent tissues. These activities favour uterine glucose uptake, decrease adipose glucose uptake, and also promote adipose mobilisation and hepatic gluconeogenesis, so as to meet the increasing energy requirement of pregnancy. It is postulated that through these effects on insulin secretion and associated adipose tissue mobilisation factors, oPL plays a major role in homeorhesis during pregnancy.
Doctor of Philosophy (PhD)

Ovine placental lactogen (oPL) is postulated to be involved in the repartitioning of maternal nutrients during pregnancy, through its effect on insulin metabolism. Ovine pancreatic insulin responses to exogenous glucose are depressed during pregnancy and this depression becomes more pronounced as gestation advances. In addition, under the hormonal environment of rising oPL and growth hormone (oGH) concentrations, maternal whole body glucose irreversible loss (GIL) increases. The percentage of GIL accounted for by uterine glucose uptake also increases with advancing gestation and increasing litter size. Regression analysis of oPL concentration with glucose uterine uptake as a percentage of GIL, accounts for 39% of variation. Maternal oPL concentrations which increase with gestational age, were significantly greater in multiple bearing ewes and ewes subjected to reduced metabolisable energy (ME) intakes. It is postulated that through actions on pancreatic sensitivity, oPL plays a major role as a homeorhetic control during pregnancy. Elevated oPL concentrations were strongly associated with continually depressed pancreatic insulin secretory ability. The reduction in pancreatic sensitivity to glucose was not as a result of elevation in GH or non-esterified fatty acid (NEFA) concentrations. Muscle insulin receptor number and affinity were found to increase with increasing litter size, suggesting that pregnancy associated insulin resistance occurs predominantly in adipose tissue. During ovine pregnancy there is a specific stimulation of maternal gluconeogenesis. As gestation advances, an increasingly greater proportion of this glucose is partitioned to the gravid uterus. The development of insulin resistance, together with the suppression of pancreatic activity, ensures the preferential uptake of glucose by non-insulin dependent tissues over insulin dependent tissues. These activities favour uterine glucose uptake, decrease adipose glucose uptake, and also promote adipose mobilisation and hepatic gluconeogenesis, so as to meet the increasing energy requirement of pregnancy. It is postulated that through these effects on insulin secretion and associated adipose tissue mobilisation factors, oPL plays a major role in homeorhesis during pregnancy.

Thesis (Ph. D. of Philosophy)--University of Western Sydney, Hawkesbury, 1997.
"A thesis submitted to the School of Agriculture and Rural Development, Faculty of Agriculture and Horticulture, University of Western Sydney Hawkesbury, in part fulfilment of the requirements for the Degree of Doctor of Philosophy." Includes bibliographical references (p. 236-267).

Prepartum, multiparous ewes were randomly assigned to experimental groups and sacrificed at 50(n=5), SO(n=4),115(n=5), and 140(n=4) days of gestation. Serum harvested the week prior to slaughter was assayed for progesterone (PG), prolactin (PRL) and growth hormone (GH) concentrations. Mammary tissue obtained at slaughter was assayed for receptor concentrations of progesterone (PGr), prolactin (PRLr) and insulin (Ir). Quantitative biochemical, histological and autoradiographical analyses were used to measure mammary gland growth and indicated no significant glandular growth occurs prior to 8O days of gestation. However, a major phase of parenchymal growth occurred between 8O and 115 days which coincided with significant increases in PG, PGr and PRLr concentrations. Parenchymal growth continued further into late pregnancy. GH and Ir concentrations did not change significantly during pregnancy and were not strongly correlated to growth measurements. These results suggest that mammary gland growth is receptor-mediated and direct or indirect regulation of PGr and PRLr is primarily responsible for the observed growth phenomenon.
Master of Science

Progesterone is unequivocally required for the establishment and maintenance of pregnancy in all mammals studied. Its known functions are complex and encompass global changes in gene expression. Therefore, studies were conducted to characterize the effects of progesterone on expression of genes for endometrial factors having roles in conceptus growth, implantation and establishment of pregnancy. The first study characterized the effect of an artificially induced early increase in circulating progesterone on conceptus growth and development and regulation of expression of galectin-15 (LGALS15), a recently identified protein secreted by the ovine uterine luminal epithelium (LE). Exogenous progesterone beginning on Day 1.5 post-mating accelerated conceptus development on Days 9 and 12. On Day 12 the conceptus was functionally and morphologically advanced to produce greater quantities of interferon tau (IFNT) than blastocysts from control ewes. Further, the endometrium responded to early progesterone and IFNT with early expression of cathepsin L (CTSL), radical S-adenosyl methionine domain containing 2 (RSAD2), and LGALS15 within the endometrium. The second study identifed structural changes within the luminal epithelium which could alter the flux of factors into and out of the uterine lumen to maintain appropriate fetal/maternal communication. In this study, progesterone reduced quantities of proteins associated with both tight and adherens junctions during the elongation period. IFNT subsequently increased these proteins after conceptus elongation. The third and fourth studies identified progesterone-regulated genes which have been implicated as having importance to implantation in sheep, mouse, and human. WNT signaling was transiently downregulated by progesterone, while members of several growth factor families are upregulated including insulin-like growth factor binding proteins (IGFBPs) 1 and 3, hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF7), which may enhance conceptus growth. Collectively, these studies assess the role of progesterone in altering gene uterine expression to establish a favorable environment for conceptus development. The long-term goals of these studies are to establish biomarkers of receptivity to conceptus development and implantation, enhance our understanding of gene and pathway regulation in early pregnancy loss, and identify genes which may be targeted in therapeutic strategies to improve reproductive success in humans and animals.

This thesis focuses on the renin-angiotensin system and atrial natriuretic factors (ANF) two hormonal systems which are stimulated and which may exert important antagonizing actions on the regulation of mean arterial pressure (MAP) and body fluid homeostasis during pregnancy. All experiments were conducted in healthy nonpregnant and/or pregnant (gestational age of 105 to 140 days, term = 145 days) mixed breed ewes, using a facility designed for studies of large animals. Findings from the first study with unilateral denervated kidneys provide evidence that renal nerves are a necessary component in the control of renin secretion in both nonpregnant and pregnant ewes. The second study demonstrates that angiotensin II (AngII) and ANF do not account for the dramatic suppression of renin secretion in response to the reduction of renal perfusion pressure in sheep with bilateral renal denervation. The data from these two studies suggest that the renal baroreceptors influence renin secretion indirectly through activation of renal afferents rather than by a direct action on the juxtaglomerular apparatus. In order to study the possible effects of increased plasma AngII on ANF production during pregnancy, four doses of AngII (0.5, 5, 20, 40 ng/kg/min) and nitroprusside were simultaneously infused to separate indirect hemodynamic actions on ANF secretion from direct hormonally mediated effects on ANF secretion by AngII. The data clearly show that AngII increases plasma ANF in a dose-dependent manner but: only in the presence of the AngII pressor effect. A striking finding was the demonstration that the natriuretic and diuretic responses to intrarenal artery infusion of three doses of ANF (0.3, 1.5, 3.0 pmol/kg/min) are increased during ovine pregnancy and these responses are solely limited to actions on the distal part of the nephron without altering renal vascular or glomerular function. Finally, a study with 10 days of intrarenal artery infusion of low dose AngII (1 ng/kg/m

Scanning and transmission electron microscopy and platelet aggregometry were employed to study erythrocyte morphology and thrombocyte morphology and function during ovine pregnancy-induced hypertension (OPIH). Nine multiparous gravid ewes were observed during normal pregnancy, OPIH, and thromboxane synthetase inhibitor administration. Blood pressure, plasma thromboxane B2, serum chemistries and electrolytes, fibrin/fibrinogen degratory products, and total platelet count were monitored throughout the investigation to document the circulatory environment during this syndrome. Arterial blood pressure, plasma thromboxane B2 levels, and serum chemistries and electrolytes were significantly altered (p≤0.005) during hypertension. However, no change in total platelet count or fibrin/fibrinogen degratory product levels were detected. Collagen-induced platelet aggregation also became abnormal during this time, while ADP-induced aggregatory response remained essentially unchanged. Platelet aggregation changes seemed to correspond to degranulation and swelling of the canalicular tubule system of these cells. Echinocytosis was present during baseline measurement and persisted throughout hypertension. However, changes in shistocyte numbers were not detected. Administration of the thromboxane synthetase inhibitors CGSl3080 or CGS12970 lowered blood pressure (p≤0.005) and serum thromboxane B₂ (p≤0.005), and. normalized serum chemistries and electrolytes (p≤0.005). Echinocyte numbers decreased (p≤0.05) and discocyte numbers increased (p≤0.005) after treatment. Platelet counts decreased after drug therapy, but normal collagen-induced aggregation was restored. No ultrastructural abnormalities were observed in thrombocytes after treatment. There is good evidence that thromboxane synthetase inhibition is appropriate and effective treatment for pregnancy-induced hypertension. These data suggest that such therapy is especially indicated in cases complicated by hematologic disorders as evidenced in an ovine model of this syndrome.
Master of Science

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum (CL) and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes including interferon stimulated genes (ISGs) that are hypothesized to be important for uterine receptivity and conceptus growth. Previous custom ovine cDNA array analyses identified candidate genes that were regulated by pregnancy, P4 and/or IFNT in the ovine uterus. The first study validated pregnancy, P4 and/or IFNT regulated genes identified by previous custom ovine cDNA microarray analyses. ACTA2, COL3A1, POSTN, SPARC, S100A2, STAT5A and TAGLN were examined. POSTN was upregulated by P4 and S100A2 was downregulated by IFNT. Moreover, functional studies showed that POSTN stimulated attachment of ovine trophectoderm cells. However, neither COL3A1, SPARC, ACTA2 nor TAGLN was regulated by pregnancy, P4 or IFNT in the ovine endometrium. Collectively, these results confirmed that POSTN and S100A2 are P4 and IFNT regulated, respectively, and likely involved in uterine receptivity to conceptus implantation during early pregnancy. The second study determined expression of ISGs in the CL of pregnant ewes. MX1, MX2, ISG15, OAS1, and RSAD2 mRNAs were increased on Day 14 of pregnancy and maintained to Day 18, indicating that IFNT induced expression of ISGs in the CL. These results confirmed that locally produced IFNT has paracrine effects and also endocrine effects on reproductive organs other than the uterine endometrium and maternal immune system.

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

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The use of artificial insemination (AI) accelerates the genetic improvement, prevents transmission of venereal diseases and facilitates testing of progeny besides enabling subfertile males to produce descendents. Fixed Time Artificial Insemination (FTAI) has been used successfully in which females have their estrus induced by hormonal treatment, which allows a prediction of the manifestation of estrus and ovulation and therefore the ideal time of insemination. In order, from the knowledge of time of ovulation, pre-determine the most appropriate period for the time of deposition of semen, cooled and frozen in the female reproductive tract in FTAI programs, twenty ewes had their estrus synchronized using intravaginal progesterone (Progespon ®, Coopers) for 12 days and 400 IU of equine chorionic gonadotropin [eCG (Novormon ®, Coopers)], to determine the moments leading up to ovulation. Other two hundred ewes, submitted to same protocol for synchronization of estrus, were divided into 4 groups for Artificial Insemination, with cooled semen used in groups 1 and 2 and frozen semen in groups 3 and 4. The sheep from G1 and G3 were inseminated 58 ± 1h after removal of progesterone intravaginal device (pre-ovulatory), and the sheeps from G2 and G4 were inseminated 64 ± 3h after withdrawal of progesterone intravaginal device (post-ovulatory). The results were obtained through the confirmation of pregnancy and data were subjected to statistical analysis with odds ratio and level of significance (p) less than 0.05. It having seen observed good results when used cooled and frozen semen, respectively, in the periods preceding (68% pregnancy rate) and exceeded (64% pregnancy) the 60h, respectively, after withdrawal of exogenous source of progestagen.
O uso da inseminação artificial acelera o melhoramento genético, evita a transmissão de doenças venéreas e facilita a realização de testes de progênie além de possibilitar que machos subférteis produzam filhos. Ultimamente, vem sendo utilizada com sucesso a inseminação artificial em tempo fixo (IATF), na qual as fêmeas têm o estro induzido por tratamento hormonal, o que permite uma previsão da manifestação do estro e ovulações e, consequentemente, o momento ideal de inseminação. Objetivando, a partir do conhecimento do momento da ovulação, pré-determinar o periodo mais adequado para o momento da deposição do sêmen, resfriado e congelado, no trato reprodutivo da fêmea, em programas de IATF, vinte ovelhas tiveram o estro sincronizado com uso de progestágenos intravaginais (Progespon®, Coopers), por 12 dias e 400UI de gonadotrofina coriônica equina [eCG (Novormon®, Coopers)], para determinação dos momentos que antecedem e marcam a ovulação. Outras duzentas ovelhas, submetidas ao mesmo protocolo para sincronização do estro, foram divididas em 4 grupos para Inseminação Artificial, tendo sido utilizado sêmen refrigerado nos grupos 1 e 2 e sêmen congelado nos grupos 3 e 4. As ovelhas que compunham os G1 e G3 foram inseminadas 58±1h após retirada do persário (pré-ovulatório), e as que compunham os G2 e G4 foram inseminadas 64±3h após retirada do persário (pós-ovulatório). Os resultados foram observados através da confirmação de prenhez e os dados submetidos à análise estatística pelo Odds ratio com e nivel de significância (p) inferior a 0,05. Tendo sido observado bons resultados quando utilizado sêmen refrigerado e congelado, respectivamente, nos períodos que antecederam (68% de prenhez) e ultrapassaram (64% de prenhez) as 60h, respectivamente, após retirada da fonte exógena de progestágeno.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
In this work, divided into three experiments, the aim was to verify the possibility to diagnose the pregnancy of does and ewes by different pathway access and diagnose the fetuses sex originated by natural mating and transfer of fresh and cryopreserved embryos. The examinations were carried out using an ultrasound scanner equipped with a linear transducer (6.0 and 8.0 MHz) used by transrectal and transabdominal via and a microconvex endocavitary (5.0 and 7.5 MHz) transducer used by transvaginal via. In the first experiment was verified the viability of the ultrasound examination by transretal, transabdominal and transvaginal via to diagnose pregnancy in goats (n = 240) and ewes (n = 320) at days 30th, 45th, 60th and 75th. In does and ewes the ultrasound examination was faster (P < 0.05) on days 30th and 45th of pregnancy, however by transabdominal via was faster on day 60th and 75th. In both species the time of pregnancy diagnose was greater (P < 0.05) on day 30th than the others days while this time was smallest (P < 0.05) at day 75th than day 45th and 60th. Independent of the examination via and the day of pregnancy the average time to diagnose the pregnancy was shorter (P < 0.05) in does than in ewes. Independing of the specie and the day of pregnancy the average time to diagnose the pregnancy by transrectal via was shorter (P < 0.05) than the other vias and the transvaginal via was shorter (P < 0.05) than the transabdominal one. In the second experiment, in order to improve the sexing of Boer fetuses (n = 123) by transrectal ultrasonography, the aim was to identify the migration period of the genital tubercle and its later differentiation into genital structures in fetuses derived from natural mating (TI) and fetus from fresh (TII), frozen (TIII) and vitrified (TIV) embryo transfer collected 7 days after breeding. Migration of the genital tubercle occurred earlier (P < 0.05) in TI (45.21 ± 3.31 days) than in TII (48.50 ± 3.70 days), TIII (48.93 ± 3.61 days) and TIV (48.85 ± 3.23 days). The visualization of the scrotum, prepuce and vulva occurred earlier (P < 0.05) in TI (51.20 ± 2.56; 50.35 ± 1.59; 49.75 ± 1.73 days) than in TII (53.25 ± 2.02; 53.37 ± 1.92; 51.76 ± 2.10 days),TIII (53.37 ± 1.92; 52.31 ± 2.00; 51 ± 78 ± 2.22 days) and TIV (54.06±1.75; 52.46 ± 1.95; 51.91 ± 2.06 days). In the third experiment, in order to improve fetal sexing by ultrasonography in Dorper ewe breed (n = 130), the objective was to identifythe migration period of the genital tubercle and the period of the visualization of external genital structures in fetuses derived from natural mating (TI) and from fresh (TII), frozen (TIII) and vitrified (TIV) embryos transfer collected 7 days after breeding. Migration of the genital tubercle occurred earlier (P < 0.05) in TI (42.21±2.86 days) than in TII (43.98±3.00 days), TIII (44.97±1.83 days) and TIV (44.58±1.97days). The visualization of scrotal bag, prepuce and vulva occurred, respectively, earlier (P < 0.05) in TI (45.22±1.25; 45.95±1.53; 45.01±.103 days) than in TII (48.91±1.92; 48.52±1.41; 47.41±1.41 days), TIII (49.97±1.08; 49.18±2.00; 47.64±1.82 days) and TIV(50.12±1.66; 49.27±1.1.61; 47.93±1.92 days). The results allow to conclude that the pregnancy diagnose may be performed by transrectal, transabdominal and transvaginal via, as well as, that is faster by transrectal via and hat the time for diagnosing is shorterin advanced pregnancy and in goat specie. Is also possible to conclude, taking into consideration the final position of the genital tubercle, that goat fetal sexing can be done from the 55th day onward in fetuses produced by natural mating and from the 60th day onward in fetuses derived from cryopreserved embryos. It can also be concluded that, real-time ultrasonography is a reliable tool for fetal sex determination in sheep after Day 50 of pregnancy taking into account both the location of the genital tubercle and the identification of external genital structures. In ovine species the fetal sexing can be done from the 50th day onward in fetuses produced by natural mating and from the 55th day onward in fetuses derived from cryopreserved embryos. Finally is possible to conclude that real-time ultrasonography is a reliable tool for early pregnancy diagnose in small ruminant as well as that to identify the sex on the first 60 days of pregnancy.
Com este trabalho, dividido em três experimentos, objetivou-se verificar a possibilidade de realizar o diagnóstico de prenhez em cabras e ovelhas por diferentes vias de acesso e sexar fetos originados de monta natural e da transferência de embriões frescos e criopreservados. Os exames foram realizados com um aparelho de ultrassom equipado com um transdutor linear (6,0 e 8,0 MHz) utilizado pelas vias transretal e transabdominal e outro microconvexo (5,0 e 7,5 MHz) endocavitário utilizado por via transvaginal. No primeiro experimento verificou-se a viabilidade do exame ultrassonográfico pelas vias transretal, transabdominal e transvaginal para diagnosticar a gestação de cabras (n = 240) e ovelhas (n = 320) no 30o, 45o, 60o e 75o dia. Nas cabras e ovelhas, o exame ultrassonográfico pela via transretal foi mais rápido (P < 0, 05) no 30o e no 45o dia da gestação, mas, pela via transabdominal foi mais rápido no 60o e no 75o dia. Em ambas as espécies, a duração do diagnóstico de gestação foi maior (P < 0,05) no 30o dia do que nos demais, enquanto que a duração do diagnóstico no 75o dia foi menor (P < 0,05) do que no 45o e 60o. Independentemente da via de exame e do dia da gestação, o tempo médio para diagnosticar a gestação foi menor (P < 0,05) nas cabras do que nas ovelhas. Independentemente da espécie e do dia da gestação, o tempo médio para diagnosticar a gestação pela via transretal foi menor (P < 0,05) do que as demais e o da transvaginal foi menor (P < 0,05) do que o da via transabdominal. No segundo experimento, com a finalidade de aperfeiçoar a sexagem de fetos (n = 123) caprinos da raça Boer por via transretal, procurou-se identificar o período de migração do tubérculo genital e sua diferenciação nas estruturas da genitália de fetos derivados de monta natural (TI) e da transferência de embriões frescos (TII), congelados (TIII) e vitrificados (TIV) colhidos 7 dias após a cobertura. A migração do tubérculo genital ocorreu mais cedo (P < 0,05) no TI (42,21±2,86 dias) do que no TII (43,98±3,00 dias), TIII (44,97±1,83 dias) e no TIV (44,58±1,97 dias). A visibilização da bolsa escrotal, prepúcio e vulva ocorreu precocemente (P < 0,05) no TI (45,22±1,25; 45,95±1,53;45,01±1,03 dias) do que no TII (53,25 ± 2,02; 53,37 ± 1,92; 51,76 ± 2,10 dias), TIII (53,25 ± 2,02; 53,37 ± 1,92; 51.76 ± 2,10 dias) e TIV (54,06±1,75; 52,46 ± 1,95;51,91 ± 2,06 dias). No terceiro experimento, com o propósito de aperfeiçoar a sexagem de fetos (n = 130) ovinos da raça Dorper por via transretal, procurou-se identificar o período de migração do tubérculo genital e sua diferenciação nas estruturas da genitália de fetos derivados de monta natural (TI) e da transferência de embriões frescos (TII), congelados (TIII) e vitrificados (TIV) colhidos 7 dias após a cobertura. A migração do tubérculo genital ocorreu mais cedo (P < 0,05) no TI (45,21 ± 3,31 dias) do que no TII (48,50 ± 3,70 dias), TIII (48.50 ± 3.70 dias) e no TIV (48,85 ± 3,23 dias). A visibilização da bolsa escrotal, prepúcio e vulva ocorreu precocemente (P < 0,05) no TI (51,20 ± 2,56; 50,35 ± 1,59; 49,75 ± 1,73 dias) do que no TII (53,25 ± 2,02; 53,37 ± 1,92; 51,76 ± 2,10 dias), TIII (53,25 ± 2,02; 53,37 ± 1,92; 51.76 ± 2,10 dias) e TIV (54,06±1,75; 52,46 ± 1,95; 51,91 ± 2,06 dias). Os resultados permitem concluir que o diagnóstico de gestação pode ser realizado pelas vias transretal, transabdominal etransvaginal, bem como que é mais rápido pela via transretal, na gestação avançada e na espécie caprina. É também permissível concluir que, com base no posicionamento final do tubérculo genital, é recomendável sexar fetos caprinos provenientes de monta natural somente a partir do 55o dia de prenhez e a partir do 60o dia naqueles derivados da transferência de embriões frescos e criopreservados. Nos ovinos a sexagem fetal já pode ser efetuada a partir do 50o dia de prenhez nos fetos oriundos de monta natural e a partir do 55o dia naqueles originados da transferência de embriões frescos e criopreservados. Ainda é possível concluir que a ultrassonografia em tempo real é uma ferramenta importante para diagnosticar precocemente a prenhez nos pequenos ruminantes, assim como identificar o sexo fetal nos primeiros 60 dias de gestação

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Universidade Estadual Paulista (UNESP)
A idade ao desmame e o estado nutricional das ovelhas no período peri-parto são aspectos determinantes para o incremento da eficiência reprodutiva e produtiva dos rebanhos ovinos. O presente trabalho teve por objetivo submeter ovelhas mestiças da raça Ile de France a dois manejos alimentares distintos (suplementadas e não suplementadas) durante o terço final da gestação e lactação, e quatro idades de desmama (60, 75, 90 e 105 dias), a fim de avaliar a variação do peso corporal e o desempenho reprodutivo das matrizes. Um total de 434 ovelhas foi avaliado quanto ao peso corporal (kg) no momento do parto (PP) e ao desmame (PD), escores de condição corporal ao parto (ECCP) e ao desmame (ECCD) e taxa de prenhez (TP). Observou-se que ovelhas suplementadas apresentaram melhor condição corporal ao parto e ao desmame (3,55 e 3,39, respectivamente) quando comparadas às não suplementadas (3,45 e 3,32, respectivamente) (P<0,05). O maior peso ao desmame observado em ovelhas suplementadas (53,2 kg) confirma um menor desgaste, pois perderam menos peso, em relação às não suplementadas (45,3 kg) (P<0,05). Durante o primeiro ano, observou-se que o manejo nutricional e a idade ao desmame influenciaram positivamente a taxa de prenhez em ovelhas que desmamaram aos 60 e 75 dias (71,88 % e 67,86 %, respectivamente) (p<0,05). Durante todo o período do ano 2 não foi observada diferença significativa na taxa de prenhez. Entretanto, o escore de condição corporal ao desmame das ovelhas do ano 1 estavam significativamente menores que o das ovelhas do ano 2 (3,30 e 3,48, respectivamente). Dessa forma, podemos indicar que a suplementação alimentar teve efeito quando as ovelhas estavam com condição corporal mais baixa, tornando-se importante a utilização de suplementação alimentar para ovelhas em terço final de gestação e durante a lactação, sendo altamente benéfica dentro do sistema de produção.

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The aim of this study, divided into two trials, was to evaluate the male effect associated with management strategies on the reproductive performance of Santa Inês ewes raised in a semi-extensive regime. In the first experiment, consisting of three treatments (T1, T2 and T3), was evaluate the effect of the separation distance between male and female, before the breeding season of 45 days, on the reproductive activity of pluriparous ewes (n = 60). They were kept at distances of 3.000 m (T1), 3 m (T2) and 300 m (T3) from the breeders (n = 3) 60 days before of the experiments during both the dry season and the rainy season. In dry season, the first manifestation of estrus averaged at 15.45 ± 10.36 (T1), 9.25 ± 6.41 (T2) and 13.05 ± 10.24 (T3) days. In rainy season, the average was 8.73 ± 5.84 (T1), 9.30 ± 5.62 (T2) and 6.10 ± 5.66 (T3) days. There was no difference in treatment (P > 0.05) during these different climatic conditions. Estrus induction occured in 100% of the females in T1, T2 and T3 in both climatic periods. Estrus synchronization on the first five days of mating season only occured in 20% of the females during dry season, being 30% (T1), 15% (T2) and 15% (T3). In rainy season, synchronisation occured in 40% of females, being 30% (T1), 35% (T2) and 45% (T3), with no differential treatment (P > 0.05) between both climatic periods. The pregnancy percentages in dry and rainy seasons were 85% (T1), 80% (T2) and 75% (T3), with no differential treatment (P > 0.05) between both climatic periods. Prolificacy in dry season was 1.29 (T1), 1.38 (T2) and 1.13 (T3) and in rainy season it was 1.12 (T1), 1.25 (T2) and 1.26 (T3) with no differential treatment (P > 0.05) between rainy and dry seasons. In the second experiment, consisting of three treatments (DT0, DT24, DT48), was evaluate the effect of temporary interrupt of sucking on the reproductive performance of pluriparous ewes (n = 60) with offspring. At DT0 (n = 20), there was no interruption of sucking while at DT24 (n = 20), interrupted of sucking for 24 hours and at DT48 (n = 20), interruption of sucking for 48 hours. Breeders were kept away from the females (n= 60), without visual, odor or hearing contact for 60 days before the breeding season of 45 days during the dry season. Estrous percentages were 90% (DT0), 100% (DT24) and 100% (DT48), with no difference (P > 0.05) among treatments. Estrous synchronization up to the 5th day of mating season was 15% (DT0), 30% (DT24) and 25% (DT48), with no difference among treatments. Pregnancy percentages were 38.4% (DT0), 60.0% (DT24) and 45.0%(DT48) with no difference (P > 0.05) among treatments. Prolificness was 1.43 (DT0), 1.17 (DT24) and 1.22 (DT48), with no difference (P > 0.05) among treatments. The results of the first experiment allow us to conclude that the ram effect can be obtained simply by avoiding physical contact between the breeder and the female as well as that the 45 day mating season can take place in both the rainy and dry season. The data from the second experiment allow us to conclude that temporary weaning is efficient to induce estrous, however is not to synchronize estrous as well as to increase the pregnancy rate and prolificacy of Santa Inês ewes in the breeding season of 45 days during dry period.
O objetivo neste estudo, dividido em dois experimentos, foi avaliar o efeito macho associado a estratégias de manejo sobre o desempenho reprodutivo de ovelhas da raça Santa Inês criadas em regime semi-extensivo. No primeiro experimento, constituído de três tratamentos (T1, T2 e T3), foi avaliado a influência da distância do afastamento entre macho e fêmea, antes da estação de monta de 45 dias, sobre o desempenho reprodutivo de fêmeas pluríparas (n = 60). As ovelhas foram afastadas por 3.000 m (T1), 3 m (T2) e 300 m (T3) de distância dos reprodutores (n = 3) 60 dias antes dos experimentos durante os períodos climáticos de seca e chuva. No período seco, o valor médio da primeira manifestação de estro foi de 15,45 ± 10,36 (T1), 9,25 ± 6,41 (T2) e de 13,05 ± 10,24 (T3) dias e no período chuvoso foi de 8,73 ± 5,84 (T1), 9,30 ± 5,62 (T2) e de 6,10 ± 5,66 (T3) dias, não havendo diferença (P > 0,05) dentro e entre tratamentos, bem como entre os diferentes períodos climáticos. A indução do estro ocorreu em 100% das fêmeas no T1, T2 e T3 em ambos os períodos climáticos. A sincronização dos estros nos primeiros cinco dias da estação de monta do período seco ocorreu somente em 20% das fêmeas, sendo de 30% (T1), 15% (T2) e de 15% (T3), não havendo diferença (P > 0,05) entre os tratamentos. No período chuvoso ocorreu em 40% das fêmeas, sendo de 30% (T1), 35% (T2) e de 45% (T3), não existindo diferença (P > 0,05) entre os tratamentos. A porcentagens de prenhez nos períodos seco e chuvoso foram de 85% (T1), 80% (T2) e de 75% (T3), não havendo diferença (P > 0,05) dentro e entre os tratamentos. A prolificidade no período seco foi de 1,29 (T1), 1,38 (T2) e de 1,13 (T3) e no período chuvoso foi de 1,12 (T1), 1,25 (T2) e de 1,26 (T3), não havendo diferença (P > 0,05) entre os tratamentos de ambos períodos climáticos. No segundo experimento, constituído de três tratamentos (DT0, DT24, DT48), foi avaliado o efeito do desaleitamento temporário associado ao efeito macho sobre o desempenho reprodutivo de ovelhas pluríparas (n = 60), com cria ao pé. No DT0 (n = 20), não houve interrupção do aleitamento, no DT24 (n = 20), o aleitamento foi interrompido por 24 horas e no DT48 (n = 20) foi interrompido por 48 horas. As ovelhas (n = 60) foram afastadas dos reprodutores (n = 3) sem contato visual, olfativo e auditivo 60 dias antes do início da estação de monta de 45 dias durante o período de seca. As porcentagens de estro foram de 90% no DT0 e de 100% no DT24 e DT48, não diferindo (P > 0,05) entre os tratamentos. A sincronização dos estros até o 5o dia da estação de monta foi de 15% (DT0), 30% (DT24) e de 25% (DT48), não havendo diferença entre os tratamentos. As porcentagens de prenhez foram de 38,4% (DT0), 60,0% (DT24) e de 45,0% (DT48), não se constatando diferença (P > 0,05) entre os tratamentos. A prolificidade foi de 1,43 (DT0), 1,17 (DT24) e de 1,22 (DT48), não havendo diferença (P > 0,05) entre os tratamentos. Os resultados do primeiro experimento permitem concluir que o efeito macho pode ser obtido evitando-se apenas o contato táctil entre reprodutor e fêmea, bem como que a estação de monta de 45 dias pode ser realizada tanto no período seco quanto no chuvoso. Com os dados do segundo experimento conclui-se que o desaleitamento temporário é eficiente para induzir o estro, mas não para sincronizá-lo e tampouco para elevar o índice de prenhez e a prolificidade das ovelhas da raça Santa Inês na estação de monta de 45 dias durante o período de seca.

Orientador: Edson Ramos de Siqueira
Banca: Ivanete Susin
Banca: João Ricardo Alves Pereira
Abstract: Weaning age and nutritional status of ewes during peri-partum period are import for increasing reproduction and production performance. The aim of this study was to investigate the effect of two different nutrition management (supplemented and unsupplemented) during late pregnancy and lactation, and four ages of weaning (60, 75, 90 and 105 days) on growth and reproductive performance of ewes. A flock of 434 Ile de France cross bred ewes was evaluated for body weight (Kg) and body condition score (ECC) at lambing (PP and ECCP) and weaning (PD and ECCD), and pregnancy rate (TP). Results showed better body condition score in supplemented group at lambing and weaning (3.55 and 3.39, respectively) than that of ewes unsupplemented (3.45 and 3.32, respectively) (p<0.05). In addition, supplemented ewes showed a higher body live weight than unsupplemented ewes (53.2 Kg and 45.3 Kg, respectively) because the supplemented ewes might not get down fast (p<0.05). During the first year of this experiment, the pregnancy rate was positively affected by nutrition and weaning age at 60 and 75 days (71.88 % and 67.86 %, respectively) (p<0.05). It was not observed significant statistical difference in pregnancy rate during the second year of this experiment. However, the body condition score of ewes at weaning in the first year was lower than that of ewes in the second year (3.30 and 3.48, respectively). Therefore, it suggests that nutrition had a great effect on ewes with low body condition. In conclusion, it is important for great results on intensive production system to supplement ewes during late gestation and weaning period.
Resumo: A idade ao desmame e o estado nutricional das ovelhas no período peri-parto são aspectos determinantes para o incremento da eficiência reprodutiva e produtiva dos rebanhos ovinos. O presente trabalho teve por objetivo submeter ovelhas mestiças da raça Ile de France a dois manejos alimentares distintos (suplementadas e não suplementadas) durante o terço final da gestação e lactação, e quatro idades de desmama (60, 75, 90 e 105 dias), a fim de avaliar a variação do peso corporal e o desempenho reprodutivo das matrizes. Um total de 434 ovelhas foi avaliado quanto ao peso corporal (kg) no momento do parto (PP) e ao desmame (PD), escores de condição corporal ao parto (ECCP) e ao desmame (ECCD) e taxa de prenhez (TP). Observou-se que ovelhas suplementadas apresentaram melhor condição corporal ao parto e ao desmame (3,55 e 3,39, respectivamente) quando comparadas às não suplementadas (3,45 e 3,32, respectivamente) (P<0,05). O maior peso ao desmame observado em ovelhas suplementadas (53,2 kg) confirma um menor desgaste, pois perderam menos peso, em relação às não suplementadas (45,3 kg) (P<0,05). Durante o primeiro ano, observou-se que o manejo nutricional e a idade ao desmame influenciaram positivamente a taxa de prenhez em ovelhas que desmamaram aos 60 e 75 dias (71,88 % e 67,86 %, respectivamente) (p<0,05). Durante todo o período do ano 2 não foi observada diferença significativa na taxa de prenhez. Entretanto, o escore de condição corporal ao desmame das ovelhas do ano 1 estavam significativamente menores que o das ovelhas do ano 2 (3,30 e 3,48, respectivamente). Dessa forma, podemos indicar que a suplementação alimentar teve efeito quando as ovelhas estavam com condição corporal mais baixa, tornando-se importante a utilização de suplementação alimentar para ovelhas em terço final de gestação e durante a lactação, sendo altamente benéfica dentro do sistema de produção.
Mestre

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It was carried out a morphological study on Oocyte Cumulus Complex of sheep (COCs), in order to characterize them quantitatively, both during the proestrus and diameter phases of the estrous cycle, as during different periods of the gestation. COCs were collected from antral follicles (3-6 mm) with regular, slightly grainy and multiple layers of cumulus cytoplasm (COC Grade I) fixed on glutaraldehyde and embedded in paraffin, as well as sectioned into sections of 5 μm. The principle of the nucleator was used to estimate average volumes of oocytes, cumulus cells and their nuclei. The thicknesses of the pellucida zone, as well as the numerical percentages of different types of cumulus cells were evaluated. There was a synchronous increase of the volume of the oocyte and its nucleus from diestrus to proestrus phases. Concerning the ones collected in the gestation stages, there was a parameter volume reduction as the pregnancy progresses. The thickness of the pellucida zone was reduced during the periods approaching ovulation. Three types of cumulus cells were observed (C1-C3). The results suggest that cumulus cells respond to circulating hormone levels during the estrous cycle and gestation.
Um estudo morfológico foi realizado em complexos cumulus oophorus de ovinos (CCOs), a fim de caracterizá-los durante as fases de proestro e dietro do ciclo estral, e durante diferentes períodos de gestação. CCOs foram colhidos a partir de folículos antrais (3-6 mm) com citoplasma regular, levemente granulado e com múltiplas camadas de células do cúmulus (CCO Grau I). Fixados em glutaraldeído e incluso em parafina, e seccionado em cortes de 5 μm. O volume médio dos oócitos com e sem zona pelúcida foram maiores nas fases de proestro, havendo redução desses volumes nas fases de diestro, G30 e G60, respectivamente. No entanto, foi observado que na fase de proestro o volume do núcleo foi inferior quando comparado aos núcleos das fases de diestro, G30 e G60. A zona pelúcida apresentou-se mais delgada à medida que no volume do oócito aumentou. As células do cumulus e seus respectivos núcleos mantiveram os volumes inalterados durante todas as fases estudadas. Houve diferença significativa quando comparado o volume dos tipos de células do cumulus em todas as fases. Também foram observados e classificados vários tipos (C1, C2 e C3) de células do cumulus, na fase de proestro houve uma maior proporção de células do tipo C3, seguido de significativa redução desse tipo de célula nas fases de diestro, G30 e G60. Já o tipo de célula C1 e C2 foram mais frequentes no grupo G60. Conclui-se que a morfologia das células do cumulus, da zona pelúcida, do citoplasma e do núcleo de complexo cumulus oophorus de folículos antrais ovinos apresenta-se morfologicamente distinta de acordo com as fases do ciclo estral e gestação.

Events around conception such as maternal undernutrition and twinning may have effects on offspring physiology and disease risk in adulthood. Periconceptional undernutrition alters offspring physiology and adult pathology without affecting birth size, while twinning affects birth size and physiology but with inconsistent effects on adult pathology. We investigated the effects of these two periconceptional events and their interaction on maternal cardiovascular adaptation to pregnancy and fetal growth, physiology and endocrinology in late gestation in sheep. Pre and/or postconception undernutrition resulted in increased uterine blood flow in late gestation, but no change in maternal blood volume. Preconception undernutrition alone resulted in a relatively large placenta with a small, slow-growing fetus in late gestation. In contrast, postconception undernutrition alone resulted in a fetus with rapid late-gestation growth that was maintained through a maternal fast. Fetuses of ewes undernourished throughout both periods were similar in growth rate and size to controls. Maternal fasting also demonstrated that plasma levels of C-type natriuretic peptide are acutely and independently regulated by nutrient supply in mother and fetus. Fetuses of ewes undernourished both pre- and postconception had increased glucose disposal following a glucose challenge. Hypothalamic-pituitary-adrenal axis tests in these fetuses showed decreased pituitary adrenocorticotropin hormone response to direct stimulation but increased adrenal response to decreased cortisol negative feedback. Twin fetuses grew more slowly in late gestation than singletons. Twins also had a smaller insulin response to arginine and a greater insulin response to glucose, but periconceptional undernutrition abolished this difference. Twins had suppressed baseline hypothalamic-pituitary-adrenal axis function and decreased adrenal sensitivity compared to singletons, but increased fetal pituitary adrenocorticotropin hormone response to direct stimulation and decreased cortisol negative feedback. These studies suggest that firstly, fetal size is a poor reflection of fetal growth trajectory, physiology and endocrinology. Secondly, pre- and postconception undernutrition affect late-gestation fetal growth in different ways, while undernutrition in both periods alters fetal endocrine status in late gestation. Thirdly, the biology of twin fetal development is fundamentally different from that of singletons, which may explain the inconsistency of the relationship between birth weight and adult disease risk in twins.
Auckland Medical Research Foundation, Health Research Council of New Zealand

Endogenous retroviruses (ERVs) comprise a significant portion of the genome of all mammals and have been implicated in placental development in multiple species. The ovine genome contains approximately 27 copies of endogenous betaretroviruses (enJSRVs) that are related to the exogenous Jaagsiekte sheep retrovirus (JSRV), an oncogenic retrovirus tropic to the lung. The enJSRV loci are abundantly expressed in the female reproductive tract and the conceptus, and they are essential to conceptus development. Studies were conducted to determine: 1) the effect of exogenous progesterone administration on conceptus development after loss of enJSRV Env; 2) the specific enJSRV env expressed in the developing conceptus; and 3) if the uterus produces enJSRV viral particles that are capable of transducing the conceptus. Study One determined the effects of exogenous progesterone on development of the conceptus in which enJSRV Env was ablated. Despite rescuing conceptus survival, the conceptuses were morphologically fragile and had reduced binucleate cell (BNC) numbers. These results suggest that mononuclear trophectoderm cell (MTC) proliferation and differentiation is dependent on enJSRV Env, even in a uterine environment supported by exogenous progesterone. Study Two assessed the enJSRV loci transcribed in the ovine conceptus during elongation before (day 13) and after (day 18) onset of BNC differentiation. The most represented loci in both day 13 and day 18 conceptuses encoded truncated Env proteins that did not contain membrane-spanning domains. Conceptuses from both time points contained evidence of the transcription of full-length, biologically active enJSRV Env, as well as completely intact proviral loci with the ability to produce viral particles in vitro. Study Three utilized a transpecies embryo transfer experiment to determine if the intact enJSRVs loci could produce viral particles in vivo. The presence of enJSRV viral particles in the uterus was confirmed, as was their ability to transduce the conceptus. Collectively, these studies provide evidence of truncated Env proteins, intact biologically active Env proteins, and enJSRVs viral particles within the ovine uterus and conceptus that are necessary to stimulate proliferation and differentiation of MTCs even in a uterine environment supported by exogenous progesterone.

Establishment of pregnancy in ruminants requires blastocyst development to form an elongated filamentous conceptus that produces interferon tau (IFNT), the pregnancy recognition signal, and initiate implantation. Blastocyst growth and development is dependent upon secretions from the uterine endometrium. An early increase in post-ovulatory circulating levels of progesterone (P4) stimulates blastocyst growth and conceptus elongation in ruminants. Microarray analysis was used to identify candidate P4-regulated genes and regulatory networks in the endometrium that govern peri-implantation blastocyst/conceptus growth and development. The first study was conducted to validate effects of P4 and/or pregnancy on expression of candidate genes identified by microarray analysis. The genes included: ANGPTL3, CHGA, CLEC4E, CXCL14, EFNA1, EFNB1, FABP3, IFNG, IL6, LGALS3, PTH, RBP4, SLIT2, SLIT3, and VWF. Early P4 treatment up-regulated CXCL14 gene expression in Day 9 ovine endometrium compared to control endometrium, and FABP3, IFNG, IL6 and LGALS3 in Day 12 early P4-treated ovine endometrium. Expression of ANGPTL3, CHGA, CXCL14, EFNA1, EFNB1, LGALS3 and RBP4 was affected by day of pregnancy. Treatment of ewes with P4+RU486 (P4 receptor antagonist) reduced expression of ANGPTL3, CHGA, EFNA1, EFNB1, FABP3, IFNG, IL6, LGALS3, RBP4, and SLIT2, SLIT3 and VWF in comparison to Day 12 P4-treated endometrium. The second study evaluated expression of genes identified by microarray analysis in endometrium from pregnant and cyclic ewes. Genes evaluated included those from the first study. ANGPTL3, CHGA, CXCL14, EFNA1, EFNB1, IFNG, LGALS3, PTH, RBP4, SLIT2, SLIT3 and VWF were affected by day, status and/or their interaction between Days 10 and 16. Of note, FABP3 increased 21-fold between Days 14 to 18 of pregnancy, and IL6 increased 37-fold between Days 14 to 20 of pregnancy. In situ hybridization analysis detected FABP3 mRNA in both luminal and superficial glandular epithelia of pregnant ewes and trophectoderm, whereas IL6 mRNA was detected in immune cells within uterine luminal epithelium and glandular epithelium and trophectoderm. Collectively, these results identify candidate genes encoding for biologically active molecules that regulate growth and development of the ovine conceptus during the peri-implantation period of pregnancy.

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.

Thesis (M.S.)--University of Wisconsin--Madison, 2004.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 133-151).