Sperm characteristics of cryopreserved Prochilodus lineatus semen after adding cholesterol-loaded cyclodextrin.

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 106 spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

Use of cholesterol-loaded cyclodextrin in donkey semen cryopreservation improves sperm viability but results in low fertility in mares.

The use of cholesterol-loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S-MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10(6) spermatozoa. Semen was frozen, and thawed samples were evaluated by computer-assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen-thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC-treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC-treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC-treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S-MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S-MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.

Progesterone and behavioral features when estrous is induced in Alpine goats.

The objective of the present study was to evaluate the endocrine and behavioral features of estrous-induced Alpine goats. A total of 36 nulliparous, 40 non-lactating and 42 lactating does were treated with intravaginal 60 mg medroxyprogesterone acetate sponges for 9 d plus 200 IU eCG and 22.5 microg d-cloprostenol 24 h before sponge removal. Plasma progesterone concentration was analyzed from blood sampled on days 0 (sponge insertion), 5, 8 (cloprostenol administration) and 9 (sponge removal) in 11 nulliparous, 13 non-lactating and 11 lactating does. Estrous response did not differ (P>0.05) among nulliparous (97.2%), non-lactating (90.00%) and lactating does (85.7%). Interval to estrus and duration of estrus did not differ (P>0.05) among nulliparous (22.8+/-9.9 and 25.6+/-6.8h), non-lactating (23.7+/-15.8 and 25.0+/-6.0 h) and lactating does (22.2+/-10.4 and 24.9+/-4.2h). The accumulative percentage of does in estrus during the first 36 h after sponge removal was 88.1%. The correlation between interval to estrus and duration of estrus was r=-0.32 (P<0.001). Endogenous progesterone production is decreased until day 8 or suppressed by MAP on day 9. Conception rate was greater (P<0.01) in lactating (77.8%) than non-lactating (44.4%) but similar (P>0.05) to nulliparous (60.0%) goats. Estrus can be efficiently induced by means of hormonal treatment in goats and acceptable fertility can be obtained regardless of animal category.

Addition of insulin-like growth factor I to the maturation medium of bovine oocytes subjected to heat shock: effects on the production of reactive oxygen species, mitochondrial activity and oocyte competence.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts.

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation.

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.

Ovarian follicular dynamic and plasma progesterone concentration in Alpine goats on the breeding season / Dinâmica folicular ovariana e concentração plasmática de progesterona em cabras Alpinas durante a estação reprodutiva

Os objetivos deste estudo foram relatar a dinâmica folicular ovariana e a concentração plasmática de progesterona durante ciclos estrais de duração irregular em cabras da raça Alpina. Vinte e nove cabras tiveram sua atividade ovariana monitorada diariamente por ultrassonografia transretal durante um, dois ou três períodos interovulatórios consecutivos. Amostras de sangue foram coletadas para análise da concentração plasmática de progesterona. Dos 47 ciclos avaliados, 28 (59,57%) foram de duração normal (17 a 25 dias), sete (14,89%) de curta duração (<17 dias) e 12 (25,53%) de longa duração (>25 dias). Os ciclos estrais de curta duração tiveram 6,14±0,69 dias e uma onda folicular que emergiu no dia 0,43±1,13. Os ciclos estrais de longa duração tiveram entre 26 e 79 dias, com duas a nove ondas foliculares. Os ciclos estrais de curta duração ocorreram durante a estação reprodutiva e foram associados à regressão prematura do corpo lúteo (CL). Os ciclos estrais de longa duração ocorreram na transição entre o anestro e a estação reprodutiva e foram associados principalmente à persistência do CL ou à fase folicular prolongada.(AU)

Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival.

This study compared the effect of adding other cholesterol conjugates, which should incorporate into and increase sperm membrane fluidity at low temperatures thereby increasing cryosurvival. Ejaculates from each of four bulls were diluted to 120 million cells/ml in a Tris diluent and used in two experiments. Each experiment contained four treatments: No additive (control); 1.5mg CLC/120 million sperm (positive control); and 1.5mg cyclodextrin pre-loaded with cholestanol or desmosterol/120 million sperm. In the first experiment, fresh sperm were treated with cyclodextrins that were pre-loaded with cholesteryl conjugates and incubated for 15 min at 22 degrees C to allow incorporation of the conjugates. The percentages of motile sperm after exposure to solutions ranging from 0 to 1200 mOsm were used to evaluate the osmotic tolerance of the cells. The ability of treated sperm to bind to the bovine zona pellucida (ZP) and chicken egg perivitelline membrane (CEPM) was evaluated to determine if altering the sperm membrane affected the ability of sperm to bind to oocytes. In the final experiment, the cryosurvival rates of control and treated sperm were determined. Control and treated sperm were cryopreserved in a Tris diluent containing 10% egg yolk (EY) and 8% glycerol (final concentrations). Samples were thawed to determine the motility and ability of sperm to bind to the ZP and to CEPM using a CASA and epifluorescence microscope, respectively. Fresh sperm treated with CLC resulted in more binding to the ZP compared to all other treatments (P<0.05). No differences were observed between ZP and CEPM binding (P>0.05). The percentages of motile sperm were greater for fresh samples treated with cholesterol, cholestanol or desmosterol loaded cyclodextrin than control cells (P<0.05) when the sperm were exposed to anisosmotic conditions, and then returned to isosmolality. After cryopreservation the percentages of motile sperm and number of sperm binding to each CEPM were similar for sperm treated with CLC and cholestanol compared to sperm treated with desmosterol (P>0.05). All treatments provided greater motility and binding efficiency than control sperm (P<0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival.

Efeito de fontes de óleo e níveis de suplementação de vitamina E na ração sobre as características físicas e morfológicas do sêmen in natura de suínos / Effect of oil sources and vitamin E supplementation in the diet on physical and morphological characteristics of fresh boar semen

Avaliou-se o efeito da adição de diferentes fontes de óleo e níveis de suplementação de vitamina E na ração sobre as características do sêmen in natura de suínos reprodutores. Foram utilizados 24 reprodutores Dalboar 85, com idades entre 12 e 18 meses, distribuídos em delineamento inteiramente ao acaso, em arranjo fatorial 2 x 3, com duas fontes de óleo, soja e salmão, e três níveis de antioxidantes, 150, 300 e 450mg de vitamina E/kg. Volume, motilidade espermática total, teste hiposmótico, porcentagem de espermatozoides vivos e morfologia espermática não diferiram (P>0,05) entre os tratamentos. Óleo de salmão (P<0,05) melhorou o vigor espermático. A inclusão de vitamina E na ração melhorou (P<0,05) a concentração espermática, e não foi observada diferença entre as fontes de óleo (P>0,05). Os animais tratados com óleo de salmão apresentaram menor (P<0,05) concentração de antioxidantes totais no sêmen do que os tratados com óleo de soja. Observou-se efeito linear da vitamina E sobre a concentração de antioxidantes totais (P<0,05). A fonte de óleo de salmão da ração melhora o vigor espermático e a concentração de antioxidantes totais no sêmen.

The addition of oil sources and dietary vitamin E supplementation was evaluated on the characteristics of fresh boar sperm. Twenty-four mature Dalboar 85 boars of proven fertility and in routine semen production for artificial insemination were randomly distributed in a factorial arrangement 2 X 3, with two oil sources, soy and salmon, and three antioxidant levels: 150, 300, and 450mg/kg of vitamin E. Volume, total motile sperm, hyposmotic swelling test, percentage of live cell, and morphology did not differ (P<0.05) between the animals from the treatments. Salmon oil improved (P<0.05) sperm vigor. The vitamin E added to the diet improved (P<0.05) the sperm concentration, and there was no difference between the oil sources (P>0.05). Salmon oil treatment showed the lowest (P<0.05) concentration of total antioxidants in the semen. Vitamin E and concentration of total antioxidant showed a linear effect (P<0.05). The semen vigor and the concentration of total antioxidants were improved by oil sources, but the sperm concentration was dependent on the levels of vitamin E. Therefore, salmon oil can be used in the diet of male pigs.

Echocardiographic diagnosis of fetal Marfan syndrome at 34 weeks' gestation.

Fetal echocardiography was performed during the third trimester in a normal primigravida. The fetal heart was severely affected with the typical cardiac manifestations of Marfan syndrome. The medical history of the father was investigated and a mild form of the syndrome was diagnosed. The neonate died at 2 months of age of congestive heart failure.

Perfil metabólico de touros da raça Nelore (Bos taurus indicus) confinados e tratados com somatotrofina bovina recombinante (r-bST) / Metabolic and hormonal profile of feedlot Nellore bulls (Bos taurus indicus) and treated with recombinant bovine somatotropin (r-bST)

Avaliaram-se os efeitos da administração da somatotrofina bovina recombinante (r-bST) sobre os metabólitos sangüíneos de touros da raça Nelore de duas diferentes idades. Foram utilizados 16 touros, distribuídos em um delineamento fatorial 2 x 2 (idades: jovens e adultos; r-bST: 0 e 500mg) com quatro animais por tratamento. A idade média dos animais foi de 13,37 e 20,62 meses para jovens e adultos, respectivamente. Quatro animais por tratamento receberam, a cada 14 dias, solução salina ou 500mg de r-bST, totalizando nove aplicações por animal, em um período experimental de 120 dias. Os touros foram alimentados com silagem de milho e ração concentrada à base de farelo de milho e soja, duas vezes por dia, fornecidas em baias individuais. As coletas de sangue foram realizadas a cada três dias, para determinação da concentração dos metabólicos sangüíneos. Para análise estatística, foram compilados dados a intervalo de três aplicações, o que constituiu um período (período 1, 2 e 3). As concentrações de ácidos graxos não-esterificados (NEFA) foram analisadas semanalmente. As concentrações séricas de colesterol, proteína total e plasmáticas de glicose diferiram para os períodos e nos grupos de tratamentos (P<0,05). As concentrações de NEFA foram influenciadas pelas semanas de coleta (P<0,05), mas não pelo tratamento com ou sem r-bST (P>0,05).

This study was carried out to evaluate the effect of recombinant bovine somatotropin (r-bST) administration on profiles of blood metabolites of two different ages Nellore bulls. Sixteen bulls were randomly allotted in a factorial arrangement 2 x 2 (ages: youngs and adults; and r-bST dose: 0 and 500 mg) with four animals per treatment. The mean ages of the young and adult animals were 13.37 and 20.62 months, respectively. Four animals per treatment received saline solution or r-bST 500mg, every 14 days, totaling nine applications per animal during 120 days. The Bulls were fed corn silage and concentrated diet based on corn crumb and soybean meal, twice a day, in individual stalls. Blood was collected every three days for metabolic evaluation. The statistical analyses of the data were performed in three applications, considering three periods (1, 2 and 3). Non-esterified fatty acid (NEFA) concentrations were weekly analyzed. Serum cholesterol, total protein and glucose levels were affected either by period or the treatment (P<0.05). The NEFA was affected by weeks of collection (P<0.05) but not by r-bST treatment (P>0.05).

Efeito do nível de uréia na dieta sobre o desempenho, a qualidade e o estádio de desenvolvimento embrionário em cabras Alpinas / Effect of the dietary urea level on performance, quality and embryonic development stage on Alpine goats

Utilizaram-se 22 cabras da raça Alpina, distribuídas aleatoriamente em quatro tratamentos (T): as cabras do T1 (n=5) formaram o grupo-controle; as do T2 (n=7) receberam 0,73 por cento de uréia na matéria seca da dieta; as do T3 (n=4) receberam 1,46 por cento de uréia; e as do T4 (n=6), 2,24 por cento de uréia. As cabras foram superovuladas e os embriões, coletados entre sete e oito dias após a primeira monta, foram avaliados quanto à qualidade e ao estádio de desenvolvimento. Amostras de sangue para dosagem dos teores de uréia e glicose foram coletadas nos dias do estro e da coleta de embriões. Houve efeito linear crescente do nível de uréia nas dietas sobre o consumo de MS (kg/dia) e de proteína bruta (kg/dia). O peso das cabras não diferiu (P>0,05) entre os tratamentos nem entre as semanas experimentais. Dezoito cabras (81,8 por cento) manifestaram estro após a sincronização. A duração do estro e o intervalo da remoção da esponja ao início do estro não foram influenciados (P>0,05) pelos tratamentos. Quatorze cabras (77,8 por cento) responderam à superovulação. O número de estruturas e de embriões coletados não diferiu (P>0,05) entre os tratamentos. O número (Y= 10,90 - 11,64NS U + 4,93§U²; R² = 0,67; P<0,10) e a percentagem (Y= 94,08 - 39,59NS U + 18,16ªU²; R² = 0,94; P<0,07) de embriões viáveis, e o número (Y= 10,83- 12,18NS U + 5,02mU²; R² = 0,78; P<0,08) e a percentagem (Y= 94,83- 52,31NS U + 21,56*U²; R² = 0,90; P<0,05) de embriões excelentes e bons apresentaram comportamento quadrático em função do nível de uréia nas dietas. Os teores de uréia e glicose no plasma não foram influenciados (P>0,05) pelos tratamentos. A uréia pode ser fornecida no nível de 2,24 por cento na MS da dieta de cabras não lactantes

Twenty-two Alpine goats were allocated at random into four treatments: 0.0 percent (T1 - control, n=5); 0.73 percent (T2, n=7); 1.46 percent (T3, n=4) or 2.24 percent (T4, n=6) of urea in the dry matter (DM) of the diet. Embryos collected from 7 to 8 days after mating of superovulated goat were evaluated by quality and development stage. Blood samples for urea and glucose analyses were collected at estrus and at embryos collection day. The DM (kg/day) and crude protein (kg/day) intake increased linearly in function of dietary urea level. Goat body weights were not affected by treatments out experimental weeks (P>0.05). Eighteen goats (81.8 percent) came in estrus after the synchronization. The estrus length and the interval from sponge removal to the beginning of estrus were not affected (P>0.05) by treatments. Fourteen goats (77.8 percent) were responsive to superovulation protocol. The levels of urea (treatments) did not affect structures and embryo numbers (P>0.05). Number (Y= 10.90 - 11.64NS U + 4.93§U²; R²= 0.67; P<0.10) and percentage (Y= 94.08 - 39.59NS U + 18.16ªU²; R²= 0.94; P<0.07) of viable embryos, and number (Y= 10.83- 12.18NS U + 5.02mU²; R²= 0.78; P<0.08) and the percentage (Y= 94.83- 52.31NS U + 21.56*U²; R²= 0.90; P<0.05) of excellent and good embryos were quadraticaly effected by urea dietary level. The treatments did not affect plasma urea and glucose levels (P>0.05). Diets for no nursing goats can be supplied by urea at 2.24 percent of DM

Sincronizacão de estro em cabras da raca Saanen com esponja intravaginal e CIDR-G® / Estrus synchonization of Saanen goats with intravaginal sponge and CIDR-G®

Compararam-se dois protocolos de curta duracão, associados à aplicacão de cipionato de estradiol (CE) para sincronizacão de estro, utilizando-se 12 cabras da raca Saanen, oito multíparas (M) e quatro nulíparas (N), distribuídas em dois tratamentos (T). O T1 (n = 4M e 2N) correspondeu à insercão de esponja impregnada com 60mg de acetato de medroxiprogesterona e o T2 (n= 4M e 2N), à insercão de CIDR-G®. No dia zero (d=0), dia da insercão dos dispositivos intravaginais, foi aplicado nos animais dos dois tratamentos 1mg de CE. Os dispositivos intravaginais permaneceram por cinco dias quando se iniciou a deteccão de estro, realizada a cada seis horas. Exames ultra-sonográficos foram feitos diariamente, durante a permanência do dispositivo, e a cada seis horas após a deteccão do estro, até 12 horas após a ovulacão. Houve regressão folicular com a emergência de uma nova onda no quinto dia da permanência do dispositivo CIDR-G®. As taxas de manifestacão de estro e de gestacão foram de 5/6 e 1/5 para T1 e de 6/6 e 2/6 para T2, respectivamente. Os intervalos da retirada do dispositivo ao início do estro e da retirada do dispositivo ao fim do estro e a duracão do estro foram: 74,0n39,1 e 34,7n15,1 horas (P<0,05), 137,2n42,1 e 90,0n11,2 horas (P<0,05) e 63,2n24,9 e 55,3n17,2 horas (P>0,05), para T1 e T2, respectivamente. Em T1 e T2, os intervalos do início do estro à ovulacão foram 50,6n22,6 e 47,3n15,3 horas (P>0,05), do final do estro à ovulacão, -12,6n9,1 e -8,0n4,5 horas (P>0,05) e da retirada do dispositivo à ovulacão, 124,6n34,1 e 82,0n12,4 horas (P<0,05), respectivamente. O número de ovulacões, o diâmetro do folículo ovulatório e a taxa de crescimento dos folículos ovulatórios foram: 1,2n0,45 e 2,33n1,03 (P<0,05), 8,4n2,4 e 6,2n0,1mm (P<0,05) e 2,4n0,97 e 2,9n1,67mm/dia (P>0,05), respectivamente, para T1 e T2. A utilizacão da esponja e do CIDR-G® associada ao CE foi efetiva em induzir o estro. A ausência de luteólise completa no momento da retirada do dispositivo foi responsável pela grande variacão nas características estudadas. Recomenda-se que os dispositivos liberadores de progesterona e seus análogos sintéticos sejam deixados por, no mínimo, sete dias, quando associados ao CE.