PLCζ, WBP2NL and TNF-α expression in spermatozoa is associated with stallion fertility and seminal quality?

This study aims to investigate the gene expression of sperm-borne phospholipase C zeta (PLCζ), WW domain-binding protein 2N-Terminal Like (WBP2NL), and Tumor necrosis factor (TNF-α), as a negative control, in spermatozoa and their relationship with fertility and seminal quality in stallions. Ejaculates from 40 Criollo stallions were used, whose fertility was assessed on the basis of their pregnancy rate per cycle in at least two breeding seasons. Pregnancy rates ranged from 20% to 90% and were used to divide the stallions into two groups: High rates (≥ 50%) (n = 25), and Low rates (< 50%) (n = 15). A computer-assisted sperm analysis system - (CASA) analyzed semen after collection. Also were evaluated the physical and functional integrity of the plasmatic membrane and sperm morphology alterations. All stallions expressed PLCζ, WBP2NL, and TNF-α. PLCζ positively correlates with conception rate, total motility (TM), progressive motility (PM), plasmatic membrane functionality, and integrity. A simple linear regression was detected between pregnancy rate and PLCζ expression (P = 0.003), TM (P < 0.001) and PM (P < 0.001). PLCζ gene expression was higher (P = 0,012) in the High rates group than in the Low group. WBP2NL and TNF-α did not correlate with seminal quality and stallion's fertility. It was concluded that PLCζ gene expression in the spermatozoa might be used as a biomarker of fertility and seminal quality in stallions. Parameters of sperm kinetics also showed, positive correlation between TM, PM and pregnancy rate.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response.

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Sperm transport and endometrial inflammatory response in mares after artificial insemination with cryopreserved spermatozoa.

This study aimed to determine whether the insemination site and dose with cryopreserved sperm of reproductively normal mares affect the sperm population in uterine tubes and the intensity of endometrial inflammatory response. Experimental subjects were estrous mares inseminated, in the mid-uterine body (Body) or the tip of the uterine horn (Tip), ipsilateral to the dominant follicle, with one 0.5 mL straw with 50 × 106 sperm (50) or with eight straws with 50 × 106 sperm/straw (400). Mares were slaughtered 2 h, 4 h and 12 h after artificial insemination (AI) and randomly assigned to following groups: Body 50 (n = 19) (2 h, 4 h or 12 h); Tip 50 (n = 29) (2 h, 4 h, or 12 h); Body 400 (n = 24) (2 h, 4 h, or 12 h); Tip 400 (n = 21) (2 h, 4 h, or 12 h). A Control group (n = 16) was not inseminated. After slaughter, uterine tubes were separated from uterus, and uteri and tubes flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected from ipsilateral and contralateral horns and mid-uterus body for further histopathological examination. A sample of each uterine tube flushing was examined for sperm count, and a sample of each uterine flushing was used for polymorphonuclear neutrophils (PMNs) count. Data were analyzed using PROC GLM from SASv9.4. Insemination time, site, sperm dose, and their interactions were considered independent variables and sperm and PMNs numbers dependent variables. Deep horn insemination increased ipsilateral uterine tube sperm number without an increase in the inflammatory reaction compared with the uterine body insemination. The higher the insemination dose, the higher the uterine tubes' sperm number and inflammatory reaction, with a quicker resolution. In conclusion, the insemination site and dose affected sperm in the uterine tubes, while post-insemination time and dose influenced the inflammatory reaction.

Hormonal Stimulation in a Gonadal Dysgenesis Mare.

The present case report aimed to determine the responsiveness of the endometrium and the ovaries of an X0 mare after hormonal treatment. On transrectal palpation, the uterus was flaccid and smaller than normal, and the ovaries were small and smooth. The endometrium had normal histological architecture, with an atrophic glandular epithelium. A karyotype evaluation was performed, and 70 cells presented 63 chromosomes, lacking one sex chromosome. Circulating hormonal levels of total estrogens were 43.93 pg/mL; progesterone 0.01 ng/mL; testosterone 48 pg/mL; FSH 30.3 ng/mL; and LH 1.71 ng/mL. Immunohistochemistry tests showed the presence of estrogens and progesterone receptors in the endometrial samples of the X0 mare. 17ß estradiol was administrated on three consecutive days and long-action progesterone on the fourth day. After hormonal stimulation, the mare showed changes in endometrial ultrasonography and histology. After treatment with estradiol, uterine edema was noted, and after progesterone, a reduction in edema was observed. At the request of the owner, no further treatment or follow-up occurred. This report showed that the endometrium is functional, but the ovaries did not change macroscopically under hormonal therapy.

Histomorphometric and vascular changes in equine endometrium after the infusion of conceptus fragments.

This experiment aimed to verify if the proteins present in a 13th day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mares daily were examined until ovulation was detected. After ovulation, mares were examined daily by transrectal palpation and B-mode and Doppler ultrasonography until day 7. On day 5, after ovulation, fragments from previously collected 13-day-old concepti were infused into the uterus of each mare. Intrauterine biopsies were collected at day 7 in all mares (n = 10), constituting the Fragment group. The percentage of ciliated and flattened cells decreased in the Fragment group. Protruded cells, superficial and intraglandular secretion, glandular lumen and diameter, blood vessel diameter, endometrial vascularization, and immune cells were higher in the Fragment group than in the Cyclic group. In summary, proteins of 13th day equine conceptus fragments infused at day five after ovulation signaled histological and vascular changes in the endometrium at the 7th day after ovulation.

Sudden Death by Ovarian Hemorrhage and Hemoperitoneum in a Pregnant Miniature Mare.

This report describes a case of sudden death of a pregnant miniature mare due to an acute ovarian hemorrhage leading to fatal hemoperitoneum. The miniature horse was a 12-year-old female, 60 days pregnant, with a body condition score of 7 (1-9), with a history of obesity and laminitis. Necropsy revealed hemoperitoneum due to an ovarian capsule rupture and hemorrhage after a physiological supplementary ovulation and luteinization. Ovarian rupture after ovulation is uncommon in mares.

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares.

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.

Ceruloplasmin, serotransferrin and albumin presented different abundance in mares' uterine fluid five days after insemination.

Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-dimensional electrophoresis (2D-PAGE). A total of 107 spots were visualized by 2D-PAGE. Three spots with differences in abundance between the inseminated and cyclic mares and with presence in at least 80% in one of the groups were selected and identified. The selected spots were extracted, digested by trypsin and analyzed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) for protein identification. Three proteins were identified: ceruloplasmin (CP) serotransferrin (TF) and albumin (ALB). The identified proteins in this study were related to iron metabolism and immunological tolerance suggesting that changes in their abundance during the 5 days after ovulation are probably a signaling mechanism of the potential equine conceptus to the maternal immune system for its immunological recognition. Probably changes in abundance of CP, ALB and TF represent a mechanism of endometrial preparation for the maternal recognition, attachment and development of a potential equine embryo. There is also evidence to support an alternative hypothesis suggesting that protein changes are inflammatory events, resulting from a previous inflammation due to residual seminal effects.

Proteomic identification of boar seminal plasma proteins related to sperm resistance to cooling at 17 °C.

The modern pig industry relies on extensive use of artificial insemination with cooled semen. It is important that semen doses maintain their quality during processing, transport and storage before insemination to guarantee maximum fertility rates. However, ejaculates may respond differently to liquid preservation at 17 °C, despite the optimal quality assessed before cooling. Thus, the aim of this study was to identify differences in seminal plasma proteome of ejaculates with a higher or lower seminal resistance to storage at 17 °C. A total of 148 ejaculates from 65 sexually mature healthy boars were classified as: High Resistance to cooling (HR, total motility > 60% at 144h) and Low resistance to cooling (LR, total motility <60 at 72h). To identify differentially expressed seminal plasma proteins between HR and LR ejaculates, ten ejaculates of each group were analyzed by 2D SDS-PAGE and ESI-Q-TOF mass spectrometry. The proteins associated with HR ejaculates were cathepsin B (spot 2803 and 6601, p < 0.01); spermadhesin PSP-I (spots 3101 and 3103, p < 0.05); epididymal secretory protein E1 precursor (spot 2101, p < 0.05) and IgGFc binding protein (spot 1603, p < 0.01). The protein associated with LR group was the Major seminal plasma PSPI (spot 9103, p < 0.01). To our knowledge, this is the first report of the association of boar seminal plasma proteins to semen resistance to cold storage at 17 °C. These results suggest the use of these proteins as biomarkers for semen resistance to preservation at 17 °C.

Effect of side of the corpus luteum and pregnancy on estrogen and progesterone receptor expression and localization in the endometrium of mares.

The effect of side of corpus luteum on uterine gene expression and protein localization of estrogen receptor α (ERα) and progesterone receptor (PR) in healthy cyclic and pregnant mares 13 days after ovulation (day 0) was investigated. Transcervical biopsies were performed to collect endometrium ipsilateral and contralateral regarding the side of corpus luteum on day 13 post-ovulation in cyclic (n = 6) and pregnant (n = 6) mares. Blood samples were collected daily from day 0 until the day of biopsy for 17ß-estradiol (E2) and progesterone (P4) determinations. Receptor expression was determined by immunohistochemistry and transcript expression by real time RT-PCR. Serum E2 and P4 concentrations were not affected by reproductive status. The contralateral horn presented higher percentage of positive cells for ERα than the ipsilateral horn (P < .05), but side did not affect PR. ERα showed low staining and no main effect of pregnancy was found, but pregnant mares had lower protein expression of PR (19.8 vs. 40.4 ±â€¯5.3%, P < .01). The contralateral horn tended to present higher expression of ERα mRNA (1.33 vs. 0.97 ±â€¯0.17, P < .10) and PR mRNA (1.96 vs. 1.57 ±â€¯0.52, P < .09). ERα mRNA relative expression was lower in the pregnant group (0.88 vs. 1.44 ±â€¯0.19, P < .05). The interaction of reproductive status and side of corpus luteum tended to affect PR mRNA expression as pregnant mares had a lower PR mRNA content in the ipsilateral horn than cyclic mares. To our knowledge, this is the first study that describes the behavior of steroid receptors in the endometrium of mares regarding side of corpus luteum.

Suplementação de touros com sabões cálcicos de ácidos graxos poli-insaturados e qualidade seminal pré e pós-congelação / Bulls supplemented with calcium soaps of polyunsaturated fatty acids and semen quality before and after freezing

O objetivo deste experimento foi avaliar a qualidade seminal de touros suplementados com sabões cálcicos de ácidos graxos poli-insaturados (PUFA). Vinte touros das raças Angus, Brangus, Hereford e Braford foram distribuídos aleatoriamente em dois grupos, cada um com dez animais. Cada grupo recebeu uma dieta composta de volumoso, ração comercial, sal mineral e um de dois suplementos: funcional com a incorporação de sabões cálcicos de PUFA ou energético, com a incorporação de raspa de mandioca (Manihot esculenta, Crantz). Durante o período experimental, a qualidade seminal foi avaliada utilizando testes rotineiros (volume, concentração, motilidade em massa, motilidade individual e avaliação da morfologia espermática) e testes complementares (teste hiposmótico, coloração dupla giemsa/tripan-azul e teste de termorresistência). O sêmen in natura do grupo de touros suplementados com sabões cálcicos de PUFA comercial (Megalac-E®) apresentou aumento na motilidade espermática (83,3% vs. 75,3%), na percentagem de espermatozoides com acrossoma íntegro (94,8% vs. 91,8%) e na percentagem de espermatozoides viáveis (98,0% vs. 96,6%) quando comparado com o sêmen do grupo de touros suplementados com raspa de mandioca (P<0,05). O tipo de suplemento não afetou o volume seminal, a concentração espermática e concentração de testosterona em sangue (P>0,05). Foram encontradas interações entre tratamento e número de coleta para percentagem de espermatozoides morfologicamente normais e percentagem de espermatozoides positivos ao teste hipo-osmótico (P<0,05). Após a descongelação, o tipo de suplemento não afetou a motilidade dos espermatozoides, o teste hipo-osmótico e o teste de termoresistência rápido (P>0,05). A suplementação com sabões cálcicos de PUFA aumentou as percentagens de espermatozoides com acrossoma íntegro (48,0% vs. 39,2%) e de espermatozoides com membrana plasmática integra (51,5% v.s 42,2%) pós-descongelação.

The objective of this experiment was to evaluate semen quality of bulls supplemented with calcium soaps of fatty acids polyunsaturated. Twenty Angus, Brangus, Hereford and Braford bulls were randomly allocated into two groups, each with ten animals. Each group received a diet consisting of forage, commercial ration, mineral supplements and supplement. Two supplements types were evaluated: functional supplement, with fatty acids polyunsaturated (PUFA) incorporation or energy supplement, with cassava meal (Manihot esculenta, Crantz) incorporation. During trial period the semen quality was evaluated using routine test (volume, concentration, mass motility, individual motility, sperm morphology) and complementary test: (hypo-osmotic test, giemsa/tripan-azul coloration and thermotolerance test). The semen of group bulls supplemented with commercial calcium soaps of PUFA (Megalac-E®) showed greater sperm motility (83.3% vs. 75.3%), percentage of sperm with intact acrosome (94.8% vs. 91.8%) and percentage of sperm with intact plasma membrane (98.0% vs. 96.6%) when compared with semen of group bulls supplemented with cassava (P<0.05). The supplement type did not affect semen volume, sperm concentration and testosterone concentration in blood (P>0.05). There were interactions between treatment and collection period for percentage of normal sperm and percentage of spermatozoa with hypo-osmotic positive test (P<0.05). The type of supplement did not affect sperm motility, the hypo-osmotic test and fast thermotolerance test after thawing (P>0.05). PUFA calcium soaps supplementation increased percentage of spermatozoa with plasma membrane integrity (51.5 vs. 42.2%) and spermatozoa intact acrosome (48.0% vs. 39.2%) after thawing.

Variação estacional na qualidade do sêmen de carneiros Lacaune no Brasil / Seasonal variation in semen quality of lacaune rams in Brazil

Little is known regarding the seasonal semen production of Lacaune rams in southern Brazil. Thus, the aim of this study was to evaluate semen quality and production of Lacaune rams maintained in Rio Grande do Sul (RS). Semen was collected using an artificial vagina, from 12 rams (1 to 4 years of age), kept under intensive conditions. The quantitative and qualitative characteristics of the semen and scrotal circumference of each ram were recorded, from the winter of 2002 to the spring of 2003. Seasonal variations were recorded in almost all the monitored semen characteristics and in scrotal circumference. Sperm concentration was the only semen characteristic evaluated that did not show seasonal variation. Sperm volume varied between 1.1 ± 0.4 mL (winter of 2002) and 1.5 ± 0.4 mL (autumn of 2003). Total number of sperm per ejaculate varied between 4.0 ± 3.3x109 (winter of 2002) and 5.7 ± 3.3x109 (summer of 2003). For scrotal circumference records, in 2002 significant differences between seasons were observed. Significant differences were also found in accordance with the age of rams (P < 0.05) in the winter of 2002. Results showed improvement in motility, concentration and volume of the sperm during summer and autumn when compared to spring and winter. So, in this region of Brazil summer and autumn are the more indicated seasons to maximize the utilization of Lacaune rams for reproductive practices. Further studies must be done to evaluate Lacaune semen freezability in these seasons of the year.

Existem poucos dados sobre a produção de sêmen da raça Lacaune, no sul do país. Assim, o objetivo deste estudo foi o de avaliar a produção e a qualidade do sêmen de carneiros desta raça, criados no estado do Rio Grande do Sul (RS). Foram utilizados 12 carneiros, de idades entre 1-4 anos, criados em condições intensivas. O sêmen foi colhido por vagina artificial e foram avaliadas as características quantitativas e qualitativas e a circunferência escrotal, do inverno de 2002 até a primavera de 2003. Com exceção da concentração espermática, foram observadas variações estacionais em todas as características estudadas. O volume variou de 1.1 ± 0.4 mL, no inverno de 2002, a 1.5 ± 0.4 mL no outono de 2003. O número total de espermatozóides por ejaculado variou de 4.0 ± 3.3x109 (inverno de 2002) a 5.7 ± 3.3x109 (verão de 2003). Observou-se variação estacional significativa (P < 0,05) na circunferência escrotal em 2002, bem como variação significativa entre as idades dos animais, no inverno do mesmo ano. Os valores médios da produção espermática observados no verão e o outono foram superiores aos obtidos no inverno e primavera. O verão e outono foram considerados as estações mais indicadas para a utilização de machos da raça Lacaune em programas reprodutivos, no RS. Mais estudos devem ser conduzidos, para a verificação de efeitos estacionais sobre o congelamento do sêmen.

Condição sociossexual de uma população de pôneis da raça Brasileira / Social-sexual condition of the Brazilian Pony Breed population

A formação dos haréns em pôneis envolve variáveis como as concentrações plasmáticas de testosterona, a idade e a dominância entre garanhões. Os objetivos deste estudo foram verificar a estabilidade e a repetibilidade da composição dos haréns e as relações entre as concentrações plasmáticas de testosterona e a condição sociossexual dos reprodutores. A estabilidade dos haréns dependeu do status reprodutivo das fêmeas. A presença de éguas gestantes no harém levou a uma maior estabilidade, enquanto que as em estro, ao procurarem outros garanhões para serem cobertas, provocaram instabilidade. A condição de dominante do garanhão foi importante para a manutenção do número de éguas no seu harém. Todos os garanhões tiveram as suas concentrações plasmáticas médias de testosterona aumentadas, em média, 77,6 por cento, quando foram expostos às éguas e quando formaram haréns (85,6 por cento), enquanto que os do grupo de solteiros tiveram as concentrações mais baixas. Portanto, fatores envolvidos na formação e estabilidade dos haréns nas populações selvagens parecem também exercer influência nas domesticadas e, consequentemente, podem interferir no desempenho destes animais durante a estação reprodutiva.

The formation of harems in ponies involves factors as testosterone level of stallion, age and dominance. The objectives were to verify the stability and repeatability of the composition of harems and the relationships between plasmatic levels of testosterone and the social-sexual condition of the stallions. The stability of harems depended of reproductive status of females and dominance of stallion. All the stallions had their mean testosterone plasmatic levels increased, in average, 77, 6 percent, when were exposed to the mares, and when they formed harems (average of 85, 6 percent), while the stallions of bachelor group had lower levels.

Ewes shorn and unshorn during pregnancy in South Brazil: effects on body condition score and lamb birth weight / Ovelhas esquiladas e não esquiladas durante a gestação no Sul do Brasil: efeito na condição corporal e no peso ao nascer dos cordeiros

In this work the effects of pre-lamb shearing on the ewe body condition scores (BCS) and lamb birth weights and growth rates were examined. A flock of 64, three year old Border Leicester x Texel ewes (49-54 kg), exposed to rams in March were divided, on day 74 of pregnancy, into two groups each of 32 ewes. The ewes grazed together, on natural pampas grass, at a stoking rate of five ewes/ha and a pasture availability of approximately 800 kg DM/ha. One group was shorn (S), at day 74 of pregnancy (P74) and the other was left unshorn (US). The BCS of the S and US ewes were assessed at the beginning of the experiment (P74), P108 and P135, as well as at lactation (L) on days L15 and L24. The lamb birth weights, and weights at 2 and 3 weeks of life were also recorded. Both groups of ewes showed a marked reduction of BCS during gestation and lactation periods. The S group experienced a significantly (P < 0.05) more severe reduction of BCS. This dropped from 3.3 score units at P74 to 1.79 and 1.22 at P135 and L24. The BCS of the US group were 3.11, 2.19 and 1.21 score units respectively at these times. Shearing pre-lambing increased mean lamb birth weight by 0.71 kg and this was statistically significant (P < 0.05). There were no effects on the lamb weight gain measured at two and three weeks of life. The increase in lamb birth weight produced by shearing ewes during pregnancy, described in this paper, could reduce the lamb perinatal mortality, especially in years with a low forage supply, leading to a critical BCS during gestation. The results suggested also that close attention should be given to the BCS of the ewe flock during pregnancy, a practice that is not common in the southern areas of Brazil.

No presente trabalho, foi testado o efeito da esquila pré-parto na condição corporal (BCS), no peso de cordeiros ao nascer e no seu desenvolvimento. O grupo experimental foi constituído de 64 ovelhas com 3 anos, cruzas Border Leicester x Texel (49-54 kg), encarneiradas em março, foram divididas, aos 74 dias de gestação, em dois grupos de 32 cada. As ovelhas foram mantidas em campo nativo, 5 ovelhas/ha, com disponibilidade aproximada de 800 kg/ha. Aos 74 dias de gestação, um grupo foi esquilado (S) e o outro foi mantido não esquilado (US). A BCS dos dois grupos foi avaliada no início do experimento (P74), P108 e P135 dias de gestação e aos 15 e 24 dias da lactação. Foi também coletado o peso dos cordeiros ao nascer e a duas e três semanas da lactação. Observou-se uma redução na BCS média das ovelhas, de ambos os grupos, durante a gestação e lactação. O grupo S experimentou uma redução significativamente mais severa (P < 0,05) na BCS. A BCS média desse grupo decresceu de 3,3 no dia P74 para 1,79 e 1,22 nos dias P135 e L24. No mesmo período, os valores da BCS média do grupo US foram de 3,11, 2,19 e 1,21. Ao nascer, o peso dos cordeiros, de ovelhas S foi 0,71 kg maior (P < 0,05) do que o dos cordeiros de ovelhas do grupo US. Não houve diferença entre o ganho de peso dos cordeiros dos dois grupos medido a duas e três semanas de vida. O aumento no peso ao nascer dos cordeiros produzido pela esquila das ovelhas durante a gestação, descrito nesse trabalho, poderá reduzir a mortalidade perinatal de cordeiros, especialmente em anos com escassez de pastagem que pode resultar em uma BCS crítica durante a gestação. Os resultados sugerem também que atenção especial deverá ser dada a BCS das ovelhas durante a gestação, prática que não é comum no Sul do Brasil.

The equine endometrosis: new insights into the pathogenesis.

This paper describes the histomorphological and immunohistochemical characterisation of phenotypic variations of endometrosis as well as potential etiological factors which may influence disease progression. In total, 779 endometrial biopsies were examined. These biopsies were taken in the breeding and non-breeding season (n=509), on defined days during the estrous cycle (n=70) and before and after experimentally induced bacterial endometritis (n=200). In addition to conventional histopathology, selected biopsies were investigated using alcianblue staining as well as immunohistochemical methods for the detection of steroid hormone receptors, Ki-67-antigen, vimentin, desmin, fibronectin, smooth-muscle-alpha-actin and laminin. The equine endometrosis can be divided into a destructive and a non-destructive form. Based on the morphology of the stromal cells involved, an active or inactive state can be distinguished in fibrotic foci. In all types of endometrosis, fibrotic stromal cells show a distinctly reduced expression of steroid hormone receptors in comparison to the intact stroma, indicating their dedifferentiation. However, the steroid hormone receptor expression of involved glandular epithelia seems to depend on the activity of the fibrosis. These results suggest an independency of all fibrotic foci from the hormonal control mechanism of the uterus. The characteristical features of destructive endometrosis are a large number of smooth-muscle-alpha-actin containing myofibroblasts, a pronounced epithelial vimentin expression, excessive extracellular matrix accumulation and a progressive alteration of the basal lamina. Furthermore, the frequently seen cystic glandular dilatation and mechanical destruction of the uterine glands may occur due to the contractibility of the myofibroblasts involved. As shown in this study, a simultaneous endometritis can cause a temporary activation of fibrotic stromal cells. However, cyclic and seasonal endocrine changes seem to have no effects on progression of the disease. It can be concluded that the various types of endometrosis represent different stages in the fibrotic process, possibly leading to the destruction of the glands and subsequently resulting in the development of a stromal fibrosis.

Criopreservação de espermatozóides eqüinos comparando duas curvas de congelamento combinadas com diluentes comerciais: uma análise laboratorial / Cryopreservation of equine spermatozoa comparing different freezing rates combined with commercial extenders: laboratorial analysis

Durante o processo de criopreservação de sêmen, os espermatozóides sofrem alguns danos que resultam na diminuição da fertilidade deste. O presente estudo foi realizado com o objetivo de avaliar os efeitos da utilização combinada de duas curvas de congelamento com dois diluentes comerciais (FR-5® e Botu-Crio®) sobre a criopreservação de sêmen eqüino. Foram analisados 20 ejaculados de dois garanhões. As amostras foram avaliadas por microscopia de contraste de fase e microscopia de epifluorescência, observando-se a motilidade progressiva e total do sêmen pós-descongelamento e a integridade e a funcionalidade da membrana dos espermatozóides. A combinação entre curva automatizada e Botu-Crio® apresentou as maiores médias nas análises de motilidade total e progressiva, após o descongelamento. O diluente Botu-Crio®, isoladamente, preservou também as membranas destes, quando foram realizadas as análises de integridade utilizando teste com diacetato de carboxifluoresceína e iodeto de propídio e funcionalidade de membrana pelo teste hiposmótico.

During semen cryopreservation, sperm cells were submitted to several deleterious events leading to membrane damage which result in fertility decrease. This study was designed to compare the effects of two freezing techniques (conventional and automated), and the use of two commercial extenders as cryoprotectants (FR-5® and Botu-Crio®) on total and progressive motility, integrity and functionality of spermatic membranes during the cryopreservation of equine semen. Twenty ejaculates from two stallions were analyzed. The total and progressive motility of fresh and post-thawing semen samples were evaluated by patterns assays. Function of plasmatic membrane was measured by the hipoosmotic swelling test. Integrity of plasmatic membrane was evaluated using carboxifluorescein diacatate and iodidium propide fluorescent probes. There were significant differences between the two freezing techniques and/or between cryoprotectants for all assessed parameters. The combination of Botu-Crio® and automated curves showed better results on total and progressive post-thawing motility. The extender Botu-Crio®, alone, showed to better preserve the membrane integrity and function.

Efeito da sincronização e da indução de estros em novilhas sobre a prenhez e o índice de repetição de crias na segunda estação reprodutiva / Estrous induction and synchronization in beef heifers and their effects on the pregnancy rate and at their performance on the second reproductive season

O objetivo deste trabalho foi verificar o índice de prenhez em novilhas de corte submetidas a um protocolo de indução e sincronização de estros sobre a cronologia das parições e seu efeito sobre a repetição de crias na estação reprodutiva subseqüente como vacas primíparas. Foram utilizadas 194 novilhas Hereford e Braford divididas em dois grupos. O grupo tratamento constou de um protocolo misto de observação e sincronização de estros e inseminação artificial a tempo fixo (IATF). O grupo controle foi submetido ao manejo convencional de inseminação artificial, com observação de estros e inseminação no turno seguinte. Ambos os grupos foram submetidos a repasse com touros. A porcentagem de prenhez na primeira estação reprodutiva para os dois grupos foi de 91,7 por cento. No grupo tratamento, 82 por cento das vacas pariram dentro dos primeiros 40 dias da estação reprodutiva, contra 51,7 por cento dos animais controle. A repetição de crias nas primíparas do grupo tratamento em comparação às do controle foi significativamente maior (73 por cento x 55 por cento). O manejo com sincronização de estros e IATF na primeira estação monta em novilhas, apesar de apresentar índices idênticos de prenhez ao final da temporada, proporcionou, porém, diferença significativa nos índices de prenhez na segunda estação reprodutiva.

This research was aimed at verifying the pregnancy rate in beef heifers submitted to a protocol for estrus induction and synchronization on the parturition period and its effect on the repetition of pregnancy at the second reproductive season. The experimental animals where composed of 194 Hereford and Braford heifers divided in two groups. The treatment group included a protocol of estrus observation and synchronization and fixed-time insemination (FTAI). The control group was submitted to a conventional artificial insemination management, with estrus observation and insemination in the next turn. Both groups were submitted to a natural mating period with clean-up bulls. The heifers were also observed as primiparous on the next reproductive season. The pregnancy rate was 91.7 percent in the first season for both groups. In the treatment group, 82 percent of the treated cows calved on the first 40 days of the groups calving season, against 51.7 percent of the control animals, resulting in a better conception rate in treated heifers (73 vs. 55 percent) in the second breeding season. The estrus synchronization and FTAI management on the first breeding season of beef heifers produce better pregnancy results also on the second breeding season.

Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare.

Our objective was to determine whether the concentration of cooled sperm inseminated influenced sperm transport and intensity of the uterine inflammatory reaction 2, 4 and 24h after insemination. Experimental subjects were 189 estrous mares with a dominant follicle > or =35 mm in diameter and no bacterial growth or neutrophils detected in uterine smears. Each mare was randomly assigned to receive one of the following intrauterine treatments (volume, 20 mL): insemination with 5x10(6) mL(-1) or 25x10(6) mL(-1) or 50x10(6) mL(-1) sperm diluted in 3 mL seminal plasma (SP) and 17 mL skim milk; seminal plasma or skim milk extender. Mares in a control group received no intrauterine treatment. Mares were slaughtered 2, 4 or 24h after insemination or infusion. Oviducts were separated from the uterus, and uterus and oviducts were then flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected for further histopathological examination. The grade of uterine fibrosis and the amount of neutrophils in the stratum compactum were evaluated. A sample of each tubal flushing was examined for sperm count, and a sample of each uterine flushing was examined for PMN count. It was concluded that compounds in the insemination dose provoked a uterine inflammatory response, which was more rapid and intense as sperm concentration increased. In contrast, sperm transport through 4h after insemination was not influenced by sperm concentration.

Use of leukocytes as treatment for endometritis in mares experimentally infected with Streptococcus equi subsp. zooepidemicus.

This study compared four treatments for bacterial endometritis in mares experimentally infected with Streptococcus zooepidemicus. Twenty-five mares were used, 20 resistant and five susceptible to endometritis. Mares would be in estrus when infected. Twenty-four hours after inoculation, clinical, bacteriological and cytological examinations were performed and repeated until the first occurrence: negative cytology and no Streptococcus growth or the seventh day post-infection. All mares showed clinical signs of endometritis and were assigned to one of the following treatments: (1) intrauterine infusion of fresh leukocytes; (2) intrauterine infusion of frozen-thawed leukocytes; (3) intrauterine infusion of lysed leukocytes; (4) intrauterine infusion of recombinant human interleukin-8 (rhIL-8); (5) control. Mares were submitted to all treatments, with at least a 14-day interval between treatments in a Latin square design. Treatment did not affect (P=0.121) time needed for resistant mares to eliminate bacteria. Time needed for elimination of bacteria was similar in susceptible mares treated with fresh and frozen leukocytes (P=0.333). Susceptible mares treated with frozen leukocytes also did not differ from those treated with lysed leukocytes (P=0.227) for time to eliminate bacteria, but were significantly different (P>0.02) from those treated with rhIL-8 and control. In resistant mares, physical clearance ability was probably the responsible for bacterial elimination. Intrauterine infusions in susceptible mares with viable or lysed leukocytes associated or not to opsonizing factors, reduced the time to elimination of bacteria. Infusions with bactericidal effect (functional neutrophils and granules) was likely effective and responsible for the more rapid elimination of bacteria in susceptible mares.

Prenhez em rebanhos ovinos do Rio Grande do Sul-Brasil / Pregnancy on sheep flocks of the state of Rio Grande do Sul-Brazil

No presente trabalho, säo apresentados dados sobre o diagnóstico de gestaçäo realizado por ultra-sonografia em 45 rebanhos ovinos, durante seis períodos reprodutivos. Foram examinadas 27.089 ovelhas de rebanhos comerciais, acasaladas durante o outono. A porcentagem de prenhez(PP) variou de 77,3 a 89,9 por cento, com uma PP média de 81,6 por cento. Rebanhos de raças de carne (Hampshire Down, Suffolk e Texel) mostram em média a PP mais elevada que foi de 85,6 por cento, seguidos de ovelhas cruzas, com PP de 82,9 por cento e finalmente ovelhas de raças de lä (Merino Australiano, Corriedale e Ideal) com PP média de 80,8 por cento. Prováveis causas de baixas PP observadas em alguns rebanhos säo apresentadas e discutidas