Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants

The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.(AU)

Three superovulation protocols for in vivo embryo production in Santa Inês sheep / Três protocolos para produção in vivo de embriões em ovelhas Santa Inês

There is still no consensus regarding the best protocol for in vivo embryo production in sheep despite increasing studies in this area. Moreover, there is variability in the response of ewes to superovulation (SOV). An approach to mitigate this inconsistency is to initiate gonadotropin administration under favorable ovarian conditions. The present study compared three treatments in a crossover design: a traditional SOV protocol (TRAD) and "Day 0" D0 SOV protocol with (D0+GnRH), or without Lecilerin (D0-GnRH). Fifteen Santa Inês ewes received 200 mg of FSH at six decreasing doses and PGF2α with the fifth dose of FSH. They were naturally mated with fertile rams and subjected to surgical embryo collection. The number of viable embryos was similar among the different treatments (TRAD = 6.0 ± 4.7; D0-GnRH = 3.8 ± 6.4; D0+GnRH = 7.5 ± 6.5). Regardless of the treatment method, ewes with follicles ≤ 4 mm, at the first FSH dose, produced more viable embryos (9.6 ± 6.0, P < 0.05) compared to ewes that had follicles > 4 mm at the beginning of the SOV (2.9 ± 3.1, viable embryos). Both the TRAD and D0+GnRH groups had fewer animals with large follicles (> 4 mm) at the first FSH dose than the D0-GnRH group (P < 0.05). In conclusion, both the TRAD and D0+GnRH treatments induced a more favorable ovarian condition (follicles ≤ 4 mm) for adequate SOV; although, all three treatments exhibited similar efficacies in Santa Inês sheep.

Ainda não há consenso sobre qual é o protocolo mais apropriado para a produção in vivo de embriões em ovinos, apesar do crescente conhecimento. Uma abordagem para mitigar a variabilidade de resposta de ovelhas à superovulação (SOV) é iniciar a aplicação de gonadotrofinas em uma condição ovariana favorável. O presente estudo comparou três tratamentos em delineamento do tipo crossover: protocolo de SOV tradicional (TRAD) e "Dia 0" D0 SOV sem (D0-GnRH) ou com GnRH (D0+GnRH). Quinze ovelhas Santa Inês foram superovuladas com 200 mg de FSH em seis doses decrescentes e receberam PGF2α na quinta dose de FSH. As ovelhas foram submetidas a monta natural com carneiros férteis e os embriões colhidos por via cirúrgica. O número de embriões viáveis não diferiu entre os tratamentos (TRAD = 6,0 ± 4,7; D0-GnRH = 3,8 ± 6,4; D0+GnRH = 7,5 ± 6,5). Independentemente do tratamento, ovelhas com folículos ≤ 4 mm na primeira dose de FSH produziram mais embriões viáveis (9,6 ± 6,0; P < 0.05) quando comparadas aos animais que apresentavam folículos > 4 mm no início da SOV (2,9 ± 3,1 embriões viáveis). Os grupos TRAD e D0+GnRH apresentaram menor número de animais com folículos grandes (> 4 mm), no momento da primeira dose de FSH, quando comparados ao grupo D0-GnRH (P < 0,05). Em conclusão, os protocolos TRAD e D0+GnRH induziram uma condição ovariana mais favorável (folículos ≤ 4 mm) para a SOV. No entanto, os três tratamentos apresentaram eficiência semelhante em ovelhas Santa Inês.

Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen / Efeito da adição de caseinato de sódio sobre a viabilidade do sêmen bubalino criopreservado

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.

O uso de sêmen resfriado em operações de inseminação artificial resulta em taxas de prenhez mais altas do que o uso de sêmen congelado. Esse resultado parece estar relacionado aos danos mais severos desencadeados pelo processo de congelação, quando comparado com o de refrigeração. Devido à sua capacidade de se ligar às proteínas ligadoras de espermatozoides e íons cálcio, o caseinato de sódio foi estudado quanto a sua capacidade de prevenir a capacitação espermática precoce, uma causa significativa de diminuição da taxa de prenhez com o uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar se um diluidor comercial a base de gema de ovo, destinado à congelação de sêmen bovino, poderia ser usado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito da adição do caseinato de sódio ao diluidor estudado durante os procedimentos de criopreservação de espermatozoides bubalinos. Foram empregadas a citometria de fluxo e a análise computadorizada do movimento espermático como métodos de avaliação seminal. Na primeira fase do estudo, comparando-se os resultados da cinética espermática e da integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação promoveu mais danos celulares que o processo de resfriamento. Na segunda fase do estudo, não foram observados efeitos benéficos da adição de caseinato de sódio ao diluente empregado. A partir dos resultados do presente estudo, foi possível concluir que o diluente à base de gema de ovo foi adequado para a criopreservação do sêmen de bubalino e que a adição do caseinato de sódio não diminuiu os efeitos deletérios desencadeados pelo processo de criopreservação seminal.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen / Efeito da adição de caseinato de sódio sobre a viabilidade do sêmen bubalino criopreservado

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.(AU)

O uso de sêmen resfriado em operações de inseminação artificial resulta em taxas de prenhez mais altas do que o uso de sêmen congelado. Esse resultado parece estar relacionado aos danos mais severos desencadeados pelo processo de congelação, quando comparado com o de refrigeração. Devido à sua capacidade de se ligar às proteínas ligadoras de espermatozoides e íons cálcio, o caseinato de sódio foi estudado quanto a sua capacidade de prevenir a capacitação espermática precoce, uma causa significativa de diminuição da taxa de prenhez com o uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar se um diluidor comercial a base de gema de ovo, destinado à congelação de sêmen bovino, poderia ser usado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito da adição do caseinato de sódio ao diluidor estudado durante os procedimentos de criopreservação de espermatozoides bubalinos. Foram empregadas a citometria de fluxo e a análise computadorizada do movimento espermático como métodos de avaliação seminal. Na primeira fase do estudo, comparando-se os resultados da cinética espermática e da integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação promoveu mais danos celulares que o processo de resfriamento. Na segunda fase do estudo, não foram observados efeitos benéficos da adição de caseinato de sódio ao diluente empregado. A partir dos resultados do presente estudo, foi possível concluir que o diluente à base de gema de ovo foi adequado para a criopreservação do sêmen de bubalino e que a adição do caseinato de sódio não diminuiu os efeitos deletérios desencadeados pelo processo de criopreservação seminal.(AU)

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P 0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.(AU)

Low density lipoproteins at 2% concentration can replace whole egg yolk in tes-tris-milk extender for freezing buffalo sperm cells

Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected.[…]

Low density lipoproteins at 2% concentration can replace whole egg yolk in tes-tris-milk extender for freezing buffalo sperm cells

Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected.[…](AU)

Peculiarities of the buffalo species for andrological evaluation – results of four years of study and weekly semen collection schedule

The aim of the present review is to provide complementary information about reproductive characteristics of male buffaloes having as perspective a four – year-study based on a weekly seminal collection schedule and collection of data in partner properties of several regions of Brazil. Aspects of testicular growth, reproductive behavior and characteristics of 1477 ejaculates of 13 donors are discussed in conjunction with data available in the literature.

Exame andrológico de bubalinos / Andrological examination of buffalos

A revisão tem como objetivo reavivar as particularidades da espécie bubalina visando a avaliaçãoandrológica. São abordadas as particularidades e anormalidades de desenvolvimento do sistema genital domacho, aspectos da puberdade e maturidade sexual, particularidades do comportamento reprodutivo,características do espermatozoide, liquido seminal e do ejaculado de búfalos.(AU)

The literature review focuses particularities of the buffalo specie and is directed to veterinarians aimingthe breeding soundness evaluation of bulls. Anatomical and abnormal developmental aspects of the genital tractare approached; puberty and sexual maturity, particularities of the reproductive behavior, characteristics of thesperm cells, seminal fluid and ejaculate are discussed.(AU)

Peculiarities of the buffalo species for andrological evaluation results of four years of study and weekly semen collection schedule

The aim of the present review is to provide complementary information about reproductive characteristics of male buffaloes having as perspective a four year-study based on a weekly seminal collection schedule and collection of data in partner properties of several regions of Brazil. Aspects of testicular growth, reproductive behavior and characteristics of 1477 ejaculates of 13 donors are discussed in conjunction with data available in the literature.(AU)

Exame andrológico de bubalinos / Andrological examination of buffalos

A revisão tem como objetivo reavivar as particularidades da espécie bubalina visando a avaliaçãoandrológica. São abordadas as particularidades e anormalidades de desenvolvimento do sistema genital domacho, aspectos da puberdade e maturidade sexual, particularidades do comportamento reprodutivo,características do espermatozoide, liquido seminal e do ejaculado de búfalos.

The literature review focuses particularities of the buffalo specie and is directed to veterinarians aimingthe breeding soundness evaluation of bulls. Anatomical and abnormal developmental aspects of the genital tractare approached; puberty and sexual maturity, particularities of the reproductive behavior, characteristics of thesperm cells, seminal fluid and ejaculate are discussed.

Effect of catalase, superoxide dismutase and reduced glutathione in LDL extender on ovine cryopreserved sperm viability / Efeito da catalase, superóxido dismutase e glutationa reduzida em diluidor contendo lipoproteínas de baixa densidade na viabilidade de espermatozoides ovino criopreservados

The aim of this study was to evaluate the motility, kinetics and membrane integrity of ovine sperm cryopreserved in extenders containing 8% LDL with enzymatic antioxidants at different concentrations. Four Santa Inês rams were used to form four pools of semen (each pool containing ejaculates from four ram, totaling four ejaculates per animal). Each seminal pool was divided into eight aliquots for the following treatments: 1) Tris-glucose-glycerol (TGG) + (16%) egg yolk (control 1); 2) TGG + 8% (w/v) LDL (control 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superoxide dismutase 100 U/mL; 6) TGG + 8% LDL + superoxide dismutase 200 U/mL; 7) TGG + 8% LDL + reduced glutathione 5 mM; and 8) TGG + 8% LDL + reduced glutathione 10 mM. The samples were packed into 0.25 mL straws, cooled (-0.25 C/ min), maintained at 5 C for 2 h and then frozen (-25 C/ min) using a TK4000®. Immediately after thawing (38 C/ 30 s), sperm motility and movement characteristics were assessed by computer sperm analysis (CASA). The structural integrity of the plasma and acrosomal membranes was analyzed using fluorescent dyes. The functional integrity of membranes was assessed using a hypoosmotic swelling test. As assessed by ANOVA, significant differences (P 0.05) among treatments were only observed for VCL, VSL and VAP. For the VCL variable, the 2, 3, 4...(AU)

Objetivou-se avaliar a motilidade, cinética e integridade das membranas de espermatozoides ovinos criopreservados em diluidores contendo 8% de LDL com antioxidantes enzimáticos em diferentes concentrações. Quatro carneiros da raça Santa Inês foram utilizados para formar quatro pools de sêmen (cada pool contendo ejaculados provenientes dos quatro carneiros, totalizando quatro ejaculados por animal). Cada pool de sêmen foi dividido em oito alíquotas para os seguintes tratamentos: 1) Tris-glicose-glicerol (TGG) + (16%) gema de ovo (controle 1); 2) TGG + 8% (g/L) LDL (controle 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superóxido dismutase 100 U/mL; 6) TGG + 8% LDL + superóxido dismutase 200 U/mL; 7) TGG + 8% LDL + glutationa reduzida 5 mM; and 8) TGG + 8% LDL + glutationa reduzida 10 mM. As amostras foram envasadas em palhetas de 0,25 mL, resfriadas (-0,25 C/min), mantidas a 5 C por duas horas e em seguida congeladas (-25 C/ min) usando uma máquina de congelar TK4000®. Imediatamente depois da descongelação (38 C/30 s), as amostras foram submetidas à análise computadorizada (CASA) para avaliação da motilidade e cinética. A integridade estrutural das membranas plasmática e acrossomal foi analisada utilizando corantes fluorescentes. A integridade funcional das membranas foi avaliada utilizando o teste hiposmótico. Como...(AU)

Low density lipoproteins added to an extender frozen or lyophilized are evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added / Lipoproteínas de baixa densidade em meio diluidor congelado ou liofilizado exerce o mesmo efeito crioprotetor aos espermatozoides ovinos que meio contendo 16% de gema de ovo

The aim of this study was to test different concentrations of low density lipoprotein (LDL) in replacement of whole egg yolk in extenders preserved in the aqueous or lyophilized form, for ram sperm cryopreservation using two cooling curves (-40C/min from 5 to 140C and nitrogen vapor). One ejaculate from six Santa Inês rams was collected. Each ejaculate was divided into nine different diluents as follows: Tris-16% yolk (control), and Tris with 2, 4, 6 and 8% fresh LDL, and criopreserved in the aqueous or lyophilized form. The samples were diluted to a final concentration of 100 x 106 sperm/mL and filled into 0.25 ml straws. After thaw, sperm cells were evaluated for motility and sperm kinetics (CASA), and submitted to the hypoosmotic swelling test and the evaluation of the structural integrity of sperm membranes using fluorescent dyes (CFDA: PI), as well as sperm morphology and longevity. The experimental design was randomized blocks, and results were submitted to ANOVA and the averages were compared using the Scott-Knott test. There were no differences in progressive motility and functional and structural integrity of the membrane evaluated when different concentrations of aqueous or lyophilized low density lipoproteins or egg yolk were added to the extender (P>0.05). As for the velocity of sperm movement, the control medium had some kinetic scores similar to the extender containing LDL, both aqueous and lyophilized (P> 0.05). Results were similar between cooling curves. Therefore, we conclude that the media containing all concentrations of LDL, aqueous or lyophilized, were able to protect the ram sperm cells during the cryopreservation process, as whole egg yolk did.(AU)

O objetivo deste trabalho foi testar para criopreservação de sêmen ovino diferentes concentrações de lipoproteína de baixa densidade (LBD) em substituição à gema de ovo em meios diluidores, armazenados na forma aquosa e liofilizada, utilizando-se duas curvas de congelação (-40C/min de 5 à 140C ou vapor de nitrogênio). Os ejaculados de seis carneiros da raça Santa Inês foram fracionados e distribuídos em nove tratamentos, sendo o primeiro o meio controle (Tris-gema 16%) e os demais meios Tris contendo lipoproteínas de baixa densidade nas concentrações de 2, 4, 6 e 8% (v/v), criopreservados tanto na forma aquosa quanto liofilizada. As amostras foram diluídas para a concentração final de 100 x 106 espermatozoides/mL e envasadas em palhetas de 0,25 mL. Após a descongelação foram avaliadas a motilidade e cinética espermática (CASA), morfologia e longevidade espermáticas, além da integridade funcional (HOST) e estrutural (CFDA/IP) das membranas. O delineamento experimental foi blocos ao acaso e os resultados foram submetidos a ANOVA, sendo as médias comparadas pelo teste de Scott-Knott. Quanto aos parâmetros de motilidade progressiva e integridade funcional e estrutural da membrana, todos os tratamentos testados foram similares (P>0,05). Quanto às velocidades do movimento espermático, o meio controle obteve alguns valores similares aos meios contendo LBD, tanto na forma aquosa quanto liofilizada (P>0,05). Não houve diferenças entre curvas de congelação. Dessa forma, é possível concluir que os meios contendo todas as concentrações de LBD, aquosa ou liofilizada, foram igualmente capazes de proteger as células espermáticas de ovinos, durante o processo de criopreservação, tanto quanto o meio contendo gema de ovo total.(AU)

Folliculogenesis in ewes: estimulation and inhibition factors of follicular growth / Foliculogênese em ovelhas: fatores de estímulo e inibição do crescimento folicular

The regulatory mechanisms of females´ reproduction are crucial to the success of conventional or assisted breeding. The follicle is the functional unit of the ovary of the female mammal species, being its major functions the growth and maturation of oocytes, as well as the production of steroid hormones. Several factors act jointly on the regulation of these activities through endocrine, autocrine and pa­racrine mechanisms stimulating the mitogenic activity of quiescent oocytes and also steroidogenic activity of the theca cells and of the granulosa. Among the regulatory factors, there are some that act in a stimulant way, such as the receptor of the binding protein (c-KITL), the system of growth factors related to insulin (IGFs), the activin and follistatin complex, and the super family of growth- transforming factor (TGF-). Other agents are inhibitory, such as inhibin, the retinoblastoma protein, expressed in oocytes, as the Wilms gene (WTI). This paper aims to review the regulatory mecha¬nisms of folliculogenesis and its importance in the reproductive physiology of ewes.

Os mecanismos de regulação da reprodução de fêmeas são de impor­tância fundamental para o sucesso da reprodução convencional ou assistida. O folículo é a unidade funcional do ovário de fêmeas das espécies mamíferas, tendo como principais funções o crescimento e a maturação dos oócitos, bem como a produção de hormônios esteróides. Vários fatores agem de forma conjunta na regulação destas atividades por meio de mecanismos endócrinos, autócrinos e parácrinos, estimulando a atividade mitogênica de oócitos quiescentes e, ativi­dade esteroidogênica de células da teca e da granulosa. Entre os fatores regu­latórios existem alguns que agem de forma estimulante, tais como o receptor da proteína ligante (c-KITL), o sistema de fatores de crescimento ligados à insulina (IGFs), o complexo ativina e folistatina e a superfamília do fator transformador de crescimento- (TGF-). Outros fatores agem de forma inibitória, tais como a inibi­na, a proteína retinoblastoma, expressa em oócitos, igualmente ao gene de Wilms (WTI). No presente trabalho, objetivou-se abordar os mecanismos regulatórios da foliculogênese e sua importância na fisiologia reprodutiva de ovelhas.

Padronização de uma técnica de congelamento de sêmen em cães

Várias pesquisas têm sido desenvolvidas com o intuito de melhorar a técnica da criopreservação de sêmen em cães. O objetivo deste trabalho foi estabelecer o meio diluidor de congelamento do sêmen canino mais adequado para um protocolo de congelamento realizado em máquina computadorizada, além de otimizar a temperatura de descongelamento. Foram usados cinco cães e coletados três ejaculados/animal. Foram testados os meios Tris-cítrico (G1), Lactose-gema (G2) e INRA 82 (G3) com 5% de glicerol. Alíquotas de sêmen foram diluídas em cada meio e foram congeladas em máquina computadorizada programada para realizar uma curva de resfriamento de -0,5ºC/min, de 25-29ºC até 5ºC, período de equilíbrio de 1h a 5ºC, seguida da curva de congelamento de -20ºC/min, de 5ºC até -120ºC. Foram testadas duas temperaturas de descongelamento: A) 37ºC/30s e B) 52ºC/10s, seguida de 37ºC/30s. Os espermatozóides foram avaliados quanto à motilidade progressiva, morfologia, reatividade ao teste hiposmótico e integridade de membranas avaliadas pelo uso de fluorescência (CFDA/IP). Os meios de congelamento Tris-cítrico e Lactose-gema foram superiores ao meio INRA quanto aos parâmetros de motilidade e morfologia espermática (p0,05). O descongelamento a 52ºC resultou no aparecimento de maior porcentagem de anormalidades acrossomais que o descongelamento a 37ºC (p

Efeito da solução, da fixação em formol-salina e do tempo de incubação sobre os resultados do teste hiposmótico para sêmen eqüino congelado

Este experimento objetivou determinar o melhor protocolo de teste hiposmótico (HO) para avaliar a integridade funcional da membrana plasmática dos espermatozóides eqüinos submetidos à criopreservação. Foram avaliadas três soluções HO (água destilada, frutose 100 mOsmol/L e citrato de sódio 100 mOsmol/L), os tempos de incubação de 0, 15 e 30 minutos dependendo do tipo de solução utilizada e o processo de fixação ou não em formol-salina. Após a descongelação do sêmen foram retiradas alíquotas de sêmen para incubação nas três soluções HO. Decorridos os tempos de incubação (0, 15 e 30 minutos), retirou-se uma alíquota de sêmen para leitura do percentual de espermatozóides reativos ao teste HO e fixou-se, em formolsalina, o restante da mistura sêmen/solução HO para posterior leitura e comparação entre as leituras fixadas versus não fixadas. Não houve diferença (P>0,05) entre os tempos de incubação independente da solução HO utilizada. A água destilada apresentou maior percentagem de espermatozóides reativos ao teste no protocolo que não usou a fixação em formol, entretanto apresentou resultado semelhante às amostras incubadas em frutose e citrato de sódio, no protocolo que utilizou a fixação em formol. Não houve diferença (P>0,05) na comparação, por solução, da leitura fixada versus não fixada. PALAVRAS-CHAVE: Eqüino, sêmen congelado, teste hiposmótico

Histomorphometry of epithelial structures of the mares endometrium

The aim of this study was to analyze through histomorphometry, epithelial structures of the mares endometrium classifiedaccording to Kenney and Doig (1986). Histomorphometric analysis of the luminal and glandular epithelial cell heights, glandularlumen diameter, glandular density, percentage of glands with apparent lumen and with intraluminal secretion, and a descriptiveanalysis of the endometrium was made in 65 endometrial biopsies. Samples were collected during estrus (n=30) and diestrus(n=35), fixed in Bouins solution, embedded in paraffin HxE and classified according to Kenney and Doig (1986) categories.Groups of IIa category obtained the greatest medium cell heights for luminal and glandular epithelium, and groups of III categoryobtained the smallest values, during estrus and diestrus. The medium heights of luminal epithelium, glandular epithelium andglandular lumen diameter of samples collected during estrus were greater than those collected during diestrus. The glanddensity was greater during diestrus than estrus. This work showed that histomorphometry could improve the consistency inbiopsy evaluation by furnishing objective data.