Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm.

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5mg CLC (positive control); 0.75 mg or 1.5mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 degrees C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 degrees C over a 2h. Upon reaching 5 degrees C, the samples were diluted 1:1 with T diluent containing 10% EY+16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686-699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments (P<0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing (P>0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control (P<0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.

The hypoosmotic swelling test in fresh rabbit spermatozoa.

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.

Sincronização de estro com CIDR reutilizado em cabras lactantes da raça Toggenburg tratadas com somatotropina bovina recombinante (r-bST) / Estrous synchronization with recycled CIDR in lactating Toggenburg does treated with recombinant bovine somatotropin (r-bST)

Avaliou-se a resposta de cabras tratadas com r-bST no protocolo de sincronização de estro. Foram utilizadas 26 cabras Toggenburg, divididas em dois tratamentos: T1 (n=13), tratadas com quatro injeções de 250mg de r-bST, a intervalos de 14 dias, e T2 (n=13), tratadas com solução salina (controle). Na semana seguinte à última injeção da r-bST, colocou-se o dispositivo intravaginal com progesterona (dia 0), previamente utilizado por cinco dias, e injetou-se PGF2alfa (22,5µg) nos animais dos dois tratamentos, e o dispositivo foi retirado no dia 6. Todas as fêmeas em estro foram submetidas à monta natural. A porcentagem de animais em estro e a taxa de gestação foram 76,9 e 70,0 e 84,6 e 72,7 por cento, no T1 e T2, respectivamente. Não houve diferença (P>0,05) na duração do estro, no intervalo tratamento-início do estro, no número de ovulações, nos intervalos: início e final do estro à ovulação e retirada do dispositivo à ovulação entre os animais dos dois tratamentos. O diâmetro médio dos folículos ovulatórios das fêmeas não diferiu (P>0,05). Durante a permanência do dispositivo, as concentrações séricas de progesterona apresentaram valores semelhantes (P>0,05) entre as cabras de T1 e T2. A r-bST não afetou a sincronização de estro.

The response of goats treated with r-bST in the estrous synchronization protocol was evaluated. Twenty-six Toggenburg females were divided in two treatments: T1 (n=13) treated with four injections of 250mg r-bST, at 14 days intervals, and T2 (n=13) treated with saline solution (control). The intravaginal device previously used by five days was inserted (day 0) one week after the last injection of r-bST and PGF2alpha (22.5µg) was injected in the animals of both treatments. The device was removed on day 6. All the females in estrus were bred by fertile bucks. The percentage of animals in estrus and the pregnancy rate were: 76.9 and 70, and 84.6 and 72.7 percent for goats in T1 and T2, respectively. The estrous cycle length; the interval from treatment to the onset of estrus; the ovulation number; and the intervals from the onset of estrus to ovulation, end of estrus to ovulation, and from device removal to ovulation were not affected by r-bST injection. The r-bST did not affect the diameter of the ovulatory follicles (P>0.05). Progesterone concentration showed similar values (P>0.05) among the goats of T1 and T2 during the treatment with device. The r-bST administration did not affect estrous synchronization.

Dinâmica folicular em cabras da raça Toggenburg em lactação tratadas ou não com somatotropina bovina recombinante / Follicular dynamics of lactating Toggenburg does treated with recombinant bovine somatotropin

Avaliou-se o efeito da injeção de somatotropina bovina recombinante (r-bST) sobre o ciclo estral de cabras. Foram utilizadas 24 fêmeas da raça Toggenburg, distribuídas em dois tratamentos: T1 (n=12), cabras tratadas com r-bST; e T2 (n=12), cabras tratadas com solução salina (controle). A partir da primeira injeção do tratamento, acompanhou-se o ciclo dos animais em estro até o estro subseqüente. O comprimento médio do ciclo estral e o período interovulatório não diferiram (P>0,05) entre os tratamentos. Foram verificados ciclos estrais de duas, três e quatro ondas de crescimento folicular. Não houve diferença (P>0,05) entre os tratamentos quanto ao número de ondas do ciclo estral. O número de folículos detectados com diâmetro >3mm nas cabras do T1 foi maior (P<0,05) que naquelas do T2 nos ciclos com duas (15,3±1,2 x 8,1±2,0), três (12,2±0,8 x 8,3±1,9) e quatro (12,7±1,7 x 8,8±2,5) ondas. O folículo dominante da segunda onda foi menor (P<0,05) que o da primeira e terceira ondas e também menor que o da onda ovulatória tanto para T1 quanto para T2. A concentração sérica de progesterona durante o ciclo estral não foi influenciada pelos tratamentos com r-bST (P>0,05). A r-bST não alterou a dinâmica folicular de cabras lactantes da raça Toggenburg, mas promoveu o aumento do número de folículos recrutados ³3mm durante o ciclo estral

The effect of the r-bST injection was evaluated on the goats estrous cycles. Twenty-four Toggenburg does were used in two treatments: T1 (n=12) treated with r-bST; and T2 (n=12) treated with saline solution (control). After the first r-bST injection, the estrous cycles were checked between two estrus. The estrous cycle length and interovulatory period of the goats did not differ between treatments (P>0.05). Estrous cycles with two, three and four waves of follicular growth were observed. The number of waves during the estrous cycle were not affected by the r-bST treatment (P>0.05). The number of 3mm follicles was different between T1 and T2 (P<0.05) for estrous cycles with two (15.3±1.2 x 8.1±2.0), three (12.2±0.8 x 8.3±1.9) or four (12.7±1.7 x 8.8±2.5) waves. The dominant follicle of the second wave was smaller (P<0.05) than the one of the first and third waves and smaller than the one of the ovulatory wave. The progesterone concentration during the estrous cycle of the animals did not differ (P>0.05) between treatments. The r-bST did not affect the ovarian response during the estrous cycle. The r-bST not affected the follicular dynamic of Toggenburg lactating does, but increased the number of emerging follicles >3mm during the estrous cycle

Indução da ovulação em cabras, fora da estação reprodutiva, com LH e GnRH e com estro induzido por progestágenos / Induction of the out-of-season ovulation (with LH and GnRH) and estrus (with progesterone) in goats

Quarenta e cinco cabras foram sincronizadas com o uso de esponjas intravaginais impregnadas com 60mg de acetato de medroxiprogesterona (MAP) por nove dias, associadas com 200UI de gonadotrofina coriônica equina (eCG) e 37,5µg de cloprostenol no sétimo dia. Os animais foram separados em três grupos e receberam, 24 horas após a retirada das esponjas, solução salina ou hormônios, no seguinte esquema: T1 (controle) - 1ml de solução salina; T2 - 5mg de hormônio luteinizante (LH) e T3 - 12,5µg do hormônio liberador de gonadotrofina (GnRH). Houve diferença (P<0,05) na porcentagem de animais em estro, 100,0 por cento, 73,3 por cento e 66,6 por cento, para T1, T2 e T3, respectivamente. Na mesma ordem, não houve diferença (P>0,05) quanto ao intervalo da retirada da esponja ao aparecimento do estro, que foi, em média, 34,8, 29,3 e 31,5 horas, respectivamente. O intervalo médio da retirada da esponja à ovulação foi de 46,6, 52,1 e 41,6 horas, respectivamente, com diferença significativa entre T2 e T3 (P<0,01). As ovulações ocorreram, em média, 21,3, 26,8 e 22,3 horas após as aplicações (P>0,05). A aplicação de LH e do GnRH para induzir e sincronizar a ovulação em cabras fora da estação reprodutiva não apresentou resultados satisfatórios.

In order to achieve estrous synchronization, intravaginal sponges impregnated with 60mg of medroxyprogesterone acetate (MPA) were implanted in 45 goats for nine days. On day 7, 200IU of equine chorionic gonadotropin (eCG) and 37.5µg of cloprostenol were administered to each doe. The animals were randomly allocated in three treatments with 15 animals each. Twenty four hours after intravaginal sponge removal, does were administered 1ml saline solution (control T1), 5mg LH (T2) and 2.5µg GnRH (T3) intramuscular injection. Percentages of animals in estrus were 100.0; 73.3 and 66.6 (P<0.05) and average intervals from sponge removal to start of estrus were 34.8, 29.3 and 31.5 hours for T1, T2 and T3, respectively, being those differences not statistically significant. The average intervals from sponge removal to ovulation were 46.6, 52.1 and 41.6 hours for T1, T2 and T3, respectively, with the difference between T2 and T3 being statistically significant (P<0.01). Ovulation time averages were 21.3, 26.8 and 22.3 hours after injections for T1, T2 and T3 , respectively.