Ultrastructural and histological characteristics of the endometrium during early embryo development in mares.

The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare's uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. From ovulation detection until day of uterine biopsy, blood samples to measure Progesterone concentrations were collected daily in cyclic and pregnant mares. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7-13. On the 7th day of pregnancy a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. The concentrations of P4 were affected by day (P < 0.001), but were not affected by group. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications may aid to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

Proteomic profile of histotroph during early embryo development in mares.

There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immediately after sample collection, the mare's uteri were flushed, and those with an embryo recovered were assigned to the Pregnant group. Of the 30 mares flushed embryos were recovered from 6 mares on day 7, 6 on day 10 and 6 on day 13. Samples from the mares without embryo recovery were excluded from both groups. The uterine fluid samples were processed by two-dimensional electrophoresis technique followed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the identification of relevant protein spots. From a total of 677 detected spots 19 were identified, 13 more abundant in Pregnant group and 6 in Cyclic group. In summary, pregnant and cyclic mares showed proteins with different abundance. Identified proteins were related to the transport of lipids through the embryo capsule, uterine motility, ATP generation, maternal immunological tolerance, cell proliferation, differentiation, metabolism and angiogenesis. Changes in the proteomic profile of uterine fluid during early embryo development in mares were related with the conceptus presence, suggesting that these alterations may be important for conceptus development and maternal recognition of pregnancy.

Metabolic effects occurring in irradiated and non-irradiated red blood cellular components for clinical transfusion practice: An in vitro comparison.

BACKGROUND: Storage lesions occur in red blood cell products when potassium ions, haemoglobin and lactate dehydrogenase are released into the extracellular plasma due to post-irradiation storage or cellular degeneration. The South African blood transfusion establishments do not comply with the universal leucocyte-reduction policy due to cost and the current HIV pandemic. Various studies regarding storage lesions have been completed in well-developed countries but not in Cape Town, South Africa. OBJECTIVE: This study aimed to determine cellular degeneration occurring in non-irradiated and irradiated red blood cells (RBC) by comparing the measured biochemical and haematological indices during storage of up to 42 days. METHOD: Eighty whole blood units were collected from voluntary, non-remunerated donors. Blood components tested weekly until expiry were whole blood, RBC concentrate, leucocyte-reduced RBC concentrate (pre-storage) and paediatric RBC concentrate (n = 20). Ten units per product were irradiated and 10 were not. Evaluations included potassium, sodium, glucose, lactate dehydrogenase, phosphate, haemoglobin, haematocrit, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentrate, mean cell volume and plasma haemoglobin. Plasma haemolysis levels were calculated using an approved formula. RESULTS: The haemolysis levels evaluated on Day 35 and Day 42 were higher than the recommended 0.8%, whereas results for the non-irradiated components up to expiry were all below 0.8%. CONCLUSION: This study confirms that gamma irradiation aggravates the RBC storage lesions. The products tested yielded similar results to other studies in developed countries, however the South Africa transfusion medicine policy should remain unchanged.

Proteomics of endometrial fluid after dexamethasone treatment in mares susceptible to endometritis.

Corticotherapy is a common treatment in mares susceptible to endometritis. Isoflupredone improves pregnancy rates and affects the protein profile of endometrial fluid in comparison to untreated mares. Dexamethasone decreases postbreeding fluid accumulation and uterine edema; however, its effects on the protein profile of the endometrial fluid have not yet been studied. The aim of the present study was to verify the effect of dexamethasone on the protein profile of endometrial fluid, in the presence or absence of infection, from mares susceptible to persistent postbreeding endometritis. Nine susceptible mares aged between 7 and 18 years were used. After checking for signs of estrus, mares were subjected to four treatments: C: mares received no treatment and served as control; D: mares received 40-mg dexamethasone at breeding, with collection of samples after 6 hours; I-6 and I-24: intrauterine infusion of 1 × 10(9)Streptococcus zooepidemicus/mL and samples collected after 6 and 24 hours; I/D-6 and I/D-24: intrauterine infusion of 1 × 10(9)S zooepidemicus/mL and 40-mg dexamethasone, collecting the sample after 6 and 24 hours. All mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis and mass spectrometry for the identification of relevant protein spots. Corticotherapy altered the protein profile of the endometrial fluid of susceptible mares, characterized by an increase and/or decrease in the optical density of inflammatory acute-phase proteins. We conclude that the use of dexamethasone in mares with and without infection alters the protein profile of endometrial fluid of susceptible mares.

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares.

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well-being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well-being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.

Resposta reprodutiva de vacas de corte associada a marcadores moleculares relacionados à fertilidade / Reproductive performance of beef cattle cows associated with molecular markers related to the fertility

O objetivo deste estudo foi buscar associação entre a taxa de prenhez após inseminação e natalidade com marcadores moleculares ligados aos genes do receptor para IGF-1, LHβ, Leptina e receptores do FSH e LH. Utilizaram-se 249 vacas adultas Aberdeen Angus, das quais 199 foram submetidas a protocolos distintos para a IATF, seguida pelo repasse com touros, e 50 vacas formaram o grupo controle representado pelo acasalamento com touros. Foram avaliados o escore de condição corporal (ECC) e o escore de condição ovariana (ECO) ao início da estação reprodutiva. O ECC influenciou a taxa de natalidade, respectivamente de 55,6%, 75,8% e 82,4% (P<0,05) para os animais com ECC menor que 2,5, entre 2,5 a 2,9, e maior ou igual a 3,0, por ocasião da estação reprodutiva. Os marcadores relacionados ao gene do receptor para o IGF-1 (AFZ-1 e HEL5) mostraram associação com a taxa de natalidade. Vacas homozigóticas para o marcador AFZ-1 apresentaram 84,4% de natalidade em comparação às heterozigóticas, com 71,5% (P<0,05). A presença do alelo*161 para o marcador HEL5 foi negativa sobre a natalidade, respectivamente de 33,3% e 76,5% para vacas com e sem esse alelo (P<0,05). Esses resultados demonstram uma importante associação entre os marcadores envolvidos com o receptor para o IGF-1 e desempenho reprodutivo de vacas Angus.

The association between the reproductive performance, expressed by pregnancy rate at fixed timed artificial insemination and birth rate in the subsequent season in beef cows, and molecular markers linked to genes for IGF-1 receptor, LHβ, leptin, and FSH and LH receptors were evaluated. Data from 249 Aberdeen Angus adult cows were used in this study. One hundred and ninety-nine cows were subjected to four different protocols for FTAI, followed by clean-up bulls and 50 cows formed the control group, matted only with bulls for 90 days during the mating season. Body condition score (BCS) and ovarian condition score (OCE) were evaluated at the beginning of the breeding season. The birth rate in the following year was 75.5%, with no treatments influence. The BCS has influenced the birth rate, respectively 55.6%, 75.8% and 82.4% (P<0.05) for animals with BCS less than 2.5; 2.5 to 2.9; and greater than or equal to 3.0, at the beginning of the breeding season. The markers related to IGF-1 receptor gene (AFZ-1 and HEL5) were associated with the birth rate in beef cows. Cows homozygous for AFZ-1 marker showed 84.4% of birth rate, while heterozygous cows showed 71.5% (P <0.05). The presence of allele *161 to the HEL5 marker was negative on birth rate. Cows with this allele had only 33.3% of birth rate, while cows without this allele had 76.5% of birth rate (P <0.05). These results demonstrate a significant association between the markers involved with the IGF-1 receptor and reproductive performance of Aberdeen Angus beef cows.

Retardo da realização da IATF sobre o desempenho reprodutivo na estação de acasalamento de vacas de corte lactantes / Delay of beginning to FTAI on reproductive performance during to mating season of beef lactating cows

Compararam-se efeitos de diferentes protocolos para a IATF com o acasalamento natural (Controle) sobre o desempenho reprodutivo de 249 vacas Aberdeen Angus, distribuídas em cinco grupos: Controle (n=50); Crestar 2º uso (n=64); OvSynch (n=65); Primer 1ºuso (n=35) e Primer 2º uso (n=35). A IATF dos animais dos grupos Crestar 2º uso, OvSynch, e Primer 1º uso foi realizada 27 dias após o início da estação do grupo controle e a IATF do grupo Primer 2º uso ocorreu 38 dias após o início da estação do grupo-controle. A partir de sete dias após a IATF, os animais foram submetidos ao repasse por touros até o término da estação de acasalamento, que foi de 91 dias para o grupo-controle, 64 dias para os grupos Crestar 2º uso, OvSynch e Primer 1º uso e de 53 dias para o grupo Primer 2º uso. A taxa de gestação ao final da estação de acasalamento não se diferenciou entre os grupos (P>0,05), sendo de 85,9%; 83,1%; 82,9%; 88,6% e 80,0%, respectivamente, para Crestar 2º uso; OvSynch; Primer 1º uso; Primer 2º uso e Controle. A taxa de parição resultante da IATF foi de 23,4%; 29,2%; 48,6% e 62,9% para os grupos Crestar 2º uso, OvSynch, Primer 1º uso, Primer 2º uso, respectivamente, com diferença significativa (P<0,05) entre Crestar e Primer 1º e 2º uso. OvSynch não se diferenciou de Crestar e Primer 1º uso. Primer 1º uso não se diferenciou de Primer 2º uso. A perda gestacional, do diagnóstico de gestação ao nascimento, foi de 10,5%. O intervalo de partos estimado (IEP) não apresentou diferenças, com média de 478 dias. O escore de condição corporal (ECC) de fêmeas gestantes ao final da estação reprodutiva foi diferente do de fêmeas não gestantes (controle), mas não dos demais grupos, possivelmente pela influência do tratamento em induzir a ciclicidade dos animais com ECC inferior. O atraso da realização da IATF após 27 ou 38 dias do início da estação de acasalamento não afetou a taxa de gestação final e o IEP dos animais, quando comparado ao acasalamento por touros.

The effects of different FTAI protocols were compared to the natural mating of bulls on the reproductive performance of 249 Aberdeen Angus cows. Five groups were formed: Control (n=50); Crestar 2nd use (n=64); OvSynch (n=65); Primer 1st use (n=35) and Primer 2nd use (n=35). The FTAI of the animals in the Crestar 2nd use, OvSynch and Primer 1st use groups was accomplished 27 days after the beginning of the mating season for the control group and the FTAI in the Primer 2nd use group happened 38 days after the beginning of the mating season of the control group. From seven days after the FTAI cows were exposed to bulls until the end of the mating season. The mating season was of 91 days for the control group, 64 days for the Crestar 2nd use, OvSynch and Primer 1st use groups and 53 days for the Primer 2nd use group. The pregnancy rate at the end of the mating season didn't differ among the groups (P>0.05), being 85.9; 83.1; 82.9; 88.6 and 80.0% respectively, for Crestar 2nd use, OvSynch, Primer 1st use, Primer 2nd use and Control. The birth rate resulting from FTAI was 23.4; 29.2; 48.6 and 62.9% for the Crestar 2nd use, OvSynch, Primer 1st use, Primer 2nd use groups, with significant difference (P<0.05) among Crestar and Primer 1st and 2nd use. OvSynch didn't differ in Crestar and Primer 1st use. Primer 1st use didn't differ from Primer 2nd use. The average reproductive losses between the gestation diagnosis and the birth were10.5%. The estimated calving interval (CI) didn't present differences among the animal groups, with an average of 478 days. The body condition score (BCS) of pregnant cows at the end of the reproductive station differed from BCS of empty cows in the control group, but it didn't differ in the other groups, possibly due to the hormonal treatment influence in inducing the oestrus and ovulation in animals with lower BCS. The delay of the accomplishment of FTAI after 27 or 38 days of the beginning of the mating season didn't affect the final pregnancy rate and CI of the cows, when compared to natural mating.

Effect of corticotherapy on proteomics of endometrial fluid from mares susceptible to persistent postbreeding endometritis.

The objective was to determine the effects of corticotherapy, in the presence and absence of uterine inflammation, on proteomics of endometrial fluid from mares susceptible to endometritis. In 11 mares, estrus was induced seven times with 5 mg PGF(2α) given at 14-day intervals. The first estrus was a control (no treatment). During the third estrus, mares received glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days. The fifth estrus was the Infected treatment (intrauterine infusion of 1 × 10(9) colony-forming unit/mL Streptococcus equi subspecies zooepidemicus). Finally, the seventh was a combination of GC + Infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected and submitted to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and mass spectrometry. Both GC treatment and uterine lumen infection induced proteomic alterations in the endometrial fluid of susceptible mares, characterized by an increase, decrease, or both in the relative optic density and/or frequency of inflammatory acute phase proteins (APP), with major alterations occurring when corticotherapy was applied in the presence of an infectious process. Corticotherapy in the presence of infection increased α(1)-antitrypsin (AAT), transthyretin (TT), and actin, but reduced immunoglobulin G, whereas intrauterine infection increased haptoglobin (Hp) and apolipoprotein A-1 (ApoA-1) and decreased transferrin (TF). Infection reduced levels of α(1)-antitrypsin and transthyretin, whereas corticotherapy in the presence of infection increased their frequency. We concluded that GC influenced the immune response, not only as suppressors, but also as enhancers of local defense mechanisms, through an immunomodulatory action. Short-term corticotherapy could be beneficial for treatment of uterine infectious processes in the mare.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.

Population pharmacokinetics of mefloquine in military personnel for prophylaxis against malaria infection during field deployment.

OBJECTIVE: The purpose of this study was to determine the population pharmacokinetics of mefloquine in healthy military personnel during prophylaxis for malaria infections. METHODS: The subjects were 1,111 Australian soldiers participating in two studies: a randomised double-blinded study (group A, 161 subjects) and an open-label study (group B, 950 subjects). Following a loading dose (250 mg mefloquine base daily, 3 days), subjects received an oral weekly maintenance dose of 250 mg over 6 months. Blood was collected after the last split loading dose then at weeks 4, 8 and 16 for group A, and at weeks 13 and 26 for group B. Plasma mefloquine concentrations were measured by high-performance liquid chromatography (HPLC). Pharmacokinetic modelling was performed using NONMEM. RESULTS: A two-compartment model with inter-occasion variability (IOV) for clearance satisfactorily described the pharmacokinetics. Typical values were clearance (CL/F, 2.09 l/h), central volume of distribution (V1/F, 528 l), absorption rate constant (KA, 0.24 h(-1)), inter-compartmental clearance (Q/F, 12.5 l/h), peripheral volume of distribution (V2/F, 483 l) and elimination half-life (t (1/2), 14.0 days). Weight had a positive influence on central volume but was insufficient to warrant dosage adjustments. The inter-individual variability (coefficient of variation, CV%) for CL/F and V1/F was 24.4% and 29.6%, respectively. The IOV for CL/F was 17.8%. The proportional residual error (CV%) for groups A and B was 11.5% and 19.5%, respectively, and the additive error standard deviation (SD) was 57 ng/ml and 149 ng/ml, respectively. CONCLUSION: The typical parameter values were comparable with those estimated in much smaller cohorts of healthy subjects and in malaria patients treated with single-dose mefloquine. The lower unexplained variability in the blinded study suggested these subjects may have been more compliant in taking their medication than soldiers in the open-label study.

Improved superovulatory response in beef cattle following ovarian follicular ablation using a simplified transvaginal device.

The influence of the timing for the ablation of dominant follicle(s) prior to superovulatory treatment, and its effect on ovarian follicular growth and embryo yield, still remain elusive in cattle. The present study was designed to evaluate the effects of: (1) the day of the estrous cycle, at mid-diestrus, for the onset of superstimulation of follicular development, (2) the presence or absence of large ovarian follicles (ovary status) and (3) the time of follicular ablation, in hours, prior to the superovulatory treatment, on the superovulatory response in cattle. From a total of 244 superovulatory treatments and embryo collections in nulliparous and multiparous females, 76 were conducted after follicular ablation using a simplified transvaginal puncture cannula. Results from the present study indicated that the presence of large palpable follicle(s) at the onset of superstimulation of follicular development markedly reduced the superovulatory response. In addition, follicular ablation at 0 h or at 24 h prior to the onset of the superstimulation treatment significantly increased the number of total viable embryos. However, superovulatory responses were not affected by the day of the estrous cycle for the onset of follicular superstimulation and by the animal category (heifers or cows). In conclusion, the ablation of palpable follicle(s) 24 h or immediately prior to the onset of gonadotropin treatment, from days 8 to 12 of the estrous cycle (day 0, behavioral estrus), increased the total number of transferable embryos per flushing in cattle.

Cerovsky: método alternativo para avaliaçäo morfológica de espermatozóides de eqüinos / Cerovsky: an alternative method for the morphological evaluation of equine spermatozoa

Foram corados 39 esfregaços de sêmen eqüino pelo método de Karras modificado e pelo método de Cerovsky. Os esfregaços foram examinados conforme preconizado por KRAUSE (1966), contando-se 200 células por lâmina. A qualidade de coloraçäo foi avaliada subjetivamente e classificada em escala de 0 a 4onde 0 corresponde à impossibilidade de leitura a 4 à ótima nitidez. Na interpretaçäo dos resultados näo foram observadas diferenças significativas (P>0,05) entre as duas técnicas. No entanto, quanto à qualidade do esfregaço, foram obtidos melhores resultados com Cerovsky. Com base nos dados obtidos conclui-se que o método de Cerovsky, além da rápida execuçäo, possibilita uma boa coloraçäo de acrossoma

The efficiency of two nonsurgical techniques for bovine embryo recovery on days 6 and 7 of the estrous cycle.

Two nonsurgical techniques for embryo recovery were employed and used in Days 6 and 7 of the estrous cycle. A significant difference between techniques was observed in both days of collection, with the closed system being more efficient in Day 6 and the opened one more efficient in Day 7. The bacteriological contamination of flushing medium (11% for the closed system and 21% for the opened technique) was of saprophytic bacteria and did not affect the percentage of pregnancies in either system.