RESUMO
INTRODUCTION: The involvement of vital organs in multiple myeloma (MM) with systemic amyloid light-chain (AL) amyloidosis can lead to acute organ failure. In this case, the fear of recurrence or progression of multiple myeloma often excludes those patients from undergoing organ transplantation. Nevertheless, clinically fit patients might benefit from a different therapeutic approach. This case presentation might highlight this particular unmet need and strengthen a different treatment approach. CASE PRESENTATION: To our knowledge, we present the first case of successful simultaneous liver and kidney transplantation, followed by autologous stem cell transplantation in a 60-year-old Caucasian male patient suffering from MM (Durie-Salmon stage IIB; ISS-stage: III, RISS stage: III) with primary AL amyloidosis. Chemotherapy treatment led to end-stage kidney disease requiring dialysis. Liver failure also occurred after at least three cycles of CyBorD (bortezomib, cyclophosphamide, and dexamethasone) of induction therapy with a good hematologic response. Over three years after the initial diagnosis, the patient is reportedly showing an excellent quality of life and a complete remission. DISCUSSION AND CONCLUSION: We conclude that kidney and liver transplantation followed by autologous stem cell transplantation can be a treatment option for a selected group of patients with MM if AL amyloidosis is leading. In the end, the remission assessment by IMWG response criteria displayed a complete remission of MM together with complete reconstitution of organ functions (liver & renal function) as long as upfront clinical evaluation excludes significant cardiac involvement and other severe co-morbidities.
Assuntos
Amiloidose , Transplante de Células-Tronco Hematopoéticas , Amiloidose de Cadeia Leve de Imunoglobulina , Transplante de Rim , Mieloma Múltiplo , Amiloidose/complicações , Amiloidose/terapia , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/complicações , Amiloidose de Cadeia Leve de Imunoglobulina/terapia , Fígado , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/complicações , Mieloma Múltiplo/terapia , Recidiva Local de Neoplasia , Qualidade de Vida , Transplante de Células-Tronco , Transplante AutólogoRESUMO
OBJECTIVES: To assess clinically relevant difference in hepatic iron quantification using R2* relaxometry with (FS) and without (non-FS) fat saturation for the evaluation of patients with suspected hepatic iron overload. METHODS: We prospectively enrolled 134 patients who underwent 1.5-T MRI R2* relaxometry with FS and non-FS gradient echo sequences (12 echoes, initial TE = 0.99 ms). Proton density fat fraction for the quantification of steatosis was assessed. Linear regression analyses and Bland-Altman plots including Lin's concordance correlation coefficient were performed for correlation of FS R2* with non-FS R2*. Patients were grouped into 4 severity classes of iron overload (EASL based), and agreement was evaluated by contingency tables and the proportion of overall agreement. RESULTS: A total of 41.8% of patients showed hepatic iron overload; 67.9% had concomitant steatosis; and 58.2% revealed no iron overload of whom 60.3% had steatosis. The mean R2* value for all FS data was 102.86 1/s, for non-FS 108.16 1/s. Linear regression resulted in an R-squared value of 0.99 (p < 0.001); Bland-Altman plot showed a mean R2* difference of 5.26 1/s (SD 17.82). The concordance correlation coefficient was only slightly lower for patients with steatosis compared with non-steatosis (0.988 vs. 0.993). The overall agreement between FS and non-FS R2* measurements was 94.8% using either method to classify patients according to severity of iron storage. No correlation between R2* and proton density fat fraction was found for both methods. CONCLUSION: R2* relaxometry showed an excellent overall agreement between FS and non-FS acquisition. Both variants can therefore be used in daily routine. However, clinically relevant differences might result when switching between the two methods or during patient follow-up, when fat content changes over time. We therefore recommend choosing a method and keeping it straight in the context of follow-up examinations. KEY POINTS: ⢠Both variants of R2* relaxometry (FS and non-FS) may be used in daily routine. ⢠Clinically relevant differences might result when switching between the two methods or during patient follow-up, when fat content changes over time. ⢠It seems advisable choosing one method and keeping it straight in the context of follow-up examinations.
Assuntos
Fígado Gorduroso/diagnóstico , Sobrecarga de Ferro/diagnóstico , Ferro/metabolismo , Fígado/metabolismo , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Feminino , Humanos , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Solutions to induce or suppress the initiation of sperm motility in fish ha ve been used to improve reproductive success during artificial fertilization and preservation techniques . The aim of the present study was to evaluate the effects of three solutions (NaCl, glucose , and BTS) - each prepared with 10 different osmolalities - on the initiation and suppression of fresh sperm motility in Prochilodus lineat us and Brycon orbignyanus . Sperm was diluted in each of the 30 solution s and immediately observed under a light microscope to determine which solution s trigger ed or suppress ed the initiation of sperm motility. When present, motility rate ( % motile sperm ) w as determined at 0, 30 , and 120 s post - activation and the motility quality score ranging from 0 ( no movement ) to 5 ( rapidly swimming sperm) was determined at 0 and 30 s post - activation . Osmolality , but not solution composition , significantly affected both motility rate and quality score . Solutions at osmolali ties up to 270 mOsm/kg in P. lineatus and up to 180 mOsm/kg in B. orbignyanus induced motility in at least 60% of sperm , with a minimum quality score of 3.0 , and were therefore classified as activating agents. The greatest motility at 0 , 30 , and 120 s post - activation was observed with solutions ranging from 135 to 225 mOsm/kg for P. lineatus and at 135 mOsm/kg for B. orbignyanus . On the other hand, solutions ranging from 360 to 450 mOsm/kg in P. lineatus and 270 to 450 mOsm/kg in B. orbignyanus suppressed motility in at least 95% of sperm and were classified as immobilizing media . The osmolality of the surrounding medium is the key factor in the initiation or suppression of sperm motility in P. lineatus a nd B. orbignyanu.
Assuntos
Animais , Motilidade dos Espermatozoides/genética , Sêmen/citologia , Peixes/classificaçãoRESUMO
Avaliou-se o quanto fêmeas e machos contribuem para a variação total das taxas de fertilização e de eclosão em curimba (Prochilodus lineatus). Utilizou-se sêmen criopreservado proveniente de cinco machos para fertilizar ovócitos de seis fêmeas em um esquema fatorial cruzado 5x6, totalizando 30 famílias. Além das características reprodutivas dos machos e fêmeas, foram avaliadas as taxas de fertilização e eclosão para cômputo dos efeitos materno e paterno. Os componentes da variância foram estimados por meio da máxima verossimilhança restrita, sendo construídos intervalos Highest Posterior Density (HPD) para cada componente. Verificou-se que as fêmeas contribuíram muito mais para a variação total em relação aos machos para as taxas de fertilização e eclosão. Para a taxa de fertilização, as fêmeas contribuíram com 26,3% da variação total e os machos com 8,9%. Em relação à taxa de eclosão, as fêmeas contribuíram com 11,9% e os machos com 1,6%. Concluiu-se que houve efeito materno sobre as taxas de fertilização e eclosão e que o efeito paterno avaliado individualmente foi pouco expressivo ou até mesmo insignificante.
The aim of this study was to evaluate how much females and males contribute to the total variation of reproductive traits such as fertilization and hatching rate in curimba Prochilodus lineatus. Cryopreserved semen from five males was used to fertilize eggs from six females in a cross-factorial 5x6, totaling 30 families. In addition to the reproductive characteristics of males and females, fertilization and hatching rates were evaluated for computation of maternal and paternal effects. The variance components were estimated by restricted maximum likelihood, and the Highest Posterior Density (HPD) intervals were estimated for each component. The female contributed more to the total variation than males for the fertilization and hatching rates. The female contributed 26.3% of the total variation in the fertilization rate against 8.9% of males. Regarding the hatching rate, the female contributed 11.9% versus 1.6% of males. Thus, there is maternal effect on rates of fertilization and hatching, and the paternal effect assessed individually was lackluster or even negligible.
Assuntos
Animais , Fertilização , Impressão Genômica , Técnicas Reprodutivas/veterinária , Criopreservação , PeixesRESUMO
Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score-arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 µm/sec of curvilinear velocity, 87 µm/sec of straight-line velocity and 103 µm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring.
Assuntos
Caraciformes/embriologia , Caraciformes/crescimento & desenvolvimento , Criopreservação , Desenvolvimento Embrionário/fisiologia , Preservação do Sêmen/efeitos adversos , Motilidade dos Espermatozoides/fisiologia , Animais , Aquicultura , Brasil , Caraciformes/fisiologia , Criopreservação/veterinária , Embrião não Mamífero , Feminino , Larva/crescimento & desenvolvimento , Masculino , Análise do Sêmen , Espermatozoides/citologia , Espermatozoides/fisiologiaRESUMO
The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide=dimethyl sulfoxide (DMSO) and methyl glycol=methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60 °C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at -170 °C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60 °C for 8 s, of regardless straw size: 74±7% motile sperm, 47±4 s of motility duration, 69±3% intact sperm, 64±4% fertilization and 63±3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.
Assuntos
Caraciformes/fisiologia , Crioprotetores/farmacologia , Congelamento , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Animais , Brasil , Ecossistema , Espécies em Perigo de Extinção , Feminino , Fertilidade , Masculino , Óvulo/fisiologia , Preservação do Sêmen/métodosRESUMO
Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 degrees C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.
Assuntos
Cocos , Criopreservação/métodos , Crioprotetores , Peixes , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Animais , Fertilidade , Fertilização/fisiologia , MasculinoRESUMO
The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P<0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality.
Assuntos
Criopreservação/veterinária , Crioprotetores , Peixes , Estações do Ano , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Criopreservação/métodos , Dimetil Sulfóxido , Glucose , Glicóis , Masculino , Rios , Preservação do Sêmen/métodos , Soluções , Motilidade dos EspermatozoidesRESUMO
Em três experimentos, avaliou-se a sensibilidade dos espermatozoides de dourado (Salminus brasiliensis) a diferentes soluções crioprotetoras. No experimento 1, o sêmen foi diluído, 1:10, em 12 soluções (quatro diluidores x três crioprotetores - dimetilsulfóxido (DMSO), metilglicol ou glicerol). Metade de cada amostra foi resfriada por uma hora e a outra, criopreservada. A motilidade espermática foi avaliada imediatamente após a diluição e após o resfriamento em todas as amostras e, após o descongelamento, apenas nas amostras criopreservadas em DMSO. No experimento 2, o sêmen foi diluído, 1:5, em cinco soluções contendo DMSO e resfriado, criopreservado e avaliado como no experimento 1. No experimento 3, o sêmen foi diluído, 1:5, em quatro soluções contendo DMSO e criopreservado e avaliado quanto à motilidade e à fertilidade. Quando o sêmen foi diluído 1:10, observou-se motilidade acima de 58 por cento em todas as amostras resfriadas em DMSO e em NaCl-tris-metilglicol. Baixa motilidade foi observada nas amostras resfriadas nas outras combinações com metilglicol (5-32 por cento) ou glicerol (0-8 por cento) e naquelas criopreservadas (16-20 por cento). Todas as amostras diluídas 1:5 apresentaram motilidade de 65-72 por cento após o resfriamento e de 45-66 por cento após o descongelamento (experimentos 2 e 3). As taxas de eclosão produzidas com sêmen criopreservado, entretanto, foram baixas (17-23 por cento) em relação ao sêmen fresco (60 por cento).
The sensitivity of dourado (Salminus brasiliensis) spermatozoa to different cryoprotectant solutions was evaluated in three experiments. In experiment 1, semen was diluted, 1:10, in 12 solutions (four extenders x three cryoprotectants - dimethylsulphoxide (DMSO), methyglycol, or glycerol). Half of each sample was refrigerated for one hour while the other half was cryopreserved. Sperm motility was immediately assessed after dilution and after refrigeration in all samples, and after thawing in those cryopreserved in DMSO. In experiment 2, semen was diluted, 1:5, in five solutions containing DMSO, refrigerated, cryopreserved, and analyzed as in experiment 1. In experiment 3, semen was diluted, 1:5, in five solutions containing DMSO, cryopreserved and evaluated for motility and fertility. When semen was diluted 1:10, motility higher than 58 percent was observed in all samples refrigerated in DMSO and in NaCl-tris-methylglycol. Low motility was observed in samples refrigerated in the other combinations of methylglycol (5-32 percent) or glycerol (0-8 percent) and in those cryopreserved (16-20 percent). All samples diluted 1:5 yielded motility of 65-72 percent after refrigeration, and 45-66 percent after thawing (experiments 2 and 3). The hatching rates produced with cryopreserved semen, however, were lower (17-23 percent) compared to fresh semen (60 percent).
RESUMO
The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.
Assuntos
Criopreservação , Peixes/fisiologia , Água Doce , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Animais , Brasil , Conservação dos Recursos Naturais , Crioprotetores , Pesqueiros , Bancos de EspermaRESUMO
The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 degrees C water bath for 8s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO(3)). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 degrees C for 8s and the other half at 30 degrees C for 16s. The 4.0-mL straws were thawed at 60 degrees C for 24s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 degrees C for 8s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.
Assuntos
Criopreservação/economia , Criopreservação/métodos , Peixes , Congelamento , Preservação do Sêmen/economia , Preservação do Sêmen/métodos , Animais , Análise Custo-Benefício , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Eficiência , Fertilização/efeitos dos fármacos , Fertilização/fisiologia , Peixes/fisiologia , Masculino , Sêmen/efeitos dos fármacos , Sêmen/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
The hypothesis was that the intramuscular injection (i.m.) of beta-carotene associated to tocopherol improves cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals with an ear implant with 3 mg of norgestomet associated with an i.m. injection of 6 mg of norgestomet and 10mg of estradiol valerate (CRESTAR, Intervert International B.V., Boxmeer, Holland) and superovulated by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h intervals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2) 800 mg of beta-carotene and 500 mg of tocopherol (T800) and (3) 1200 mg of beta-carotene and 750 mg of tocopherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at first superovulatory injection. An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo quality (excellent*1 + good*2 + regular*3 + poor*4 + degenerate*5 + unfertilized ova*5)/total. There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6+/-0.3 and 3.6+/-0.3; 2.5+/-0.3 and 3.6+/-0.3; 2.9+/-0.3 and 2.7+/-0.3, respectively. The average total number of viable embryos was greater (P=0.01) in supplemented cows (3.5+/-1.1; 5.4+/-1.4 and 7.5+/-1.2 in control, T800 and T1200, respectively), but less (P=0.01) in heifers (7.5+/-1.2; 5.6+/-1.2 and 4.0+/-1.1 in control, T800 and T1200, respectively). Supplementation injections of beta-carotene associated to tocopherol improved embryo quality in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar embryo production systems. However, at dosages applied in the present experiment, this treatment should not be recommended for nulliparous heifers.
Assuntos
Suplementos Nutricionais , Embrião de Mamíferos/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Tocoferóis/farmacologia , beta Caroteno/farmacologia , Animais , Bovinos , Contagem de Células , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização/efeitos dos fármacos , Fertilização/fisiologia , Ovulação/fisiologia , Gravidez , Taxa de Gravidez , Controle de Qualidade , Superovulação/efeitos dos fármacosRESUMO
Avaliaram-se protocolos de resfriamento e de criopreservação do sêmen de pirapitinga (Brycon nattereri) utilizando-se sêmen diluído em NaCl 154mM, NaCl 200mM, Saad e BTS® e resfriado por sete dias. Cinco diluidores (glicose 277mM, NaCl 154mM, NaCl 200mM, Saad e BTS®) foram combinados com dois crioprotetores (DMSO - dimetilsulfóxido e metilglicol) e usados como meio de congelamento. O sêmen diluído em cada meio foi envasado (palhetas de 0,5ml) e congelado, e a motilidade espermática avaliada após o descongelamento (60ºC, 8seg). O sêmen foi novamente congelado em palhetas com diferentes volumes (0,25 e 0,5ml) e descongelados em banho-maria em duas temperaturas (50º e 60ºC). As maiores motilidades (48 por cento) foram observadas no sêmen diluído em BTS® e resfriado por sete dias. Motilidade espermática acima de 68 por cento foram observadas no sêmen congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO. Não houve diferença entre os volumes de palheta nem entre as temperaturas de descongelamento quanto a motilidade espermática. Assim, o sêmen de pirapitinga mantém altas taxas de motilidade quando resfriado em BTS® por até sete dias ou congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO
Cooling and freezing protocols of pirapitinga (Brycon nattereri) semen were evaluated using semen diluted in 154mM NaCl, 200mM NaCl, Saad or BTSÕ, and cooled for seven days. Sperm motility was daily evaluated. Five extenders (277mM glucose, 154mM NaCl, 200mM NaCl, Saad and BTSÕ) were combined with two cryoprotectants (DMSO - dimethyl sulphoxide and methylglycol) to produce 10 cryosolutions. Semen was diluted in each cryosolutions, aspirated into 0.5ml straws and frozen. Sperm motility was evaluated after thawing (60ºC, 8 sec). Then, semen was frozen in straws with different volumes (0.25 and 0.5ml), and thawed under different water-bath temperatures (50º and 60ºC). Higher sperm motility (48 percent) was observed when semen was cooled in BTSÕ for seven days. Post-thawing sperm motility above 68 percent was observed when semen was frozen in 154mM NaCl-methylglycol, BTSÕ-methylglycol, 200mM NaCl-DMSO or Saad-DMSO. There was no difference on sperm motility when semen was frozen in 0.25 or 0.5ml straws and thawed in 50º or 60ºC water-bath. Thus, pirapitinga semen can be successfully cooled in BTSÕ for seven days or frozen in 154 mM NaCl-methylglycol, BTSÕ- methylglycol, 200mM NaCl-DMSO and Saad-DMSO
Assuntos
Animais , Masculino , Peixes , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Criopreservação/métodos , Criopreservação/veterináriaRESUMO
In silurid fishes, semen collection is practically impossible, even after hormonal stimulation. Instead, males are killed and testes macerated to obtain sperm. To understand the endocrine control of semen release in catfishes, we investigated the role of smooth muscle contractors in semen release and semen quality of African catfish (Clarias gariepinus). In in vitro experiments, testis slices were incubated with oxytocin (1 and 10 IU), isotocin (2 and 20 ug), vasopressin (0.2 and 2 ug), epinephrine (1 and 10 ug), PGF2alpha (1 and 10 ug), purified Clarias LH (300 ng) and partly purified Clarias pituitary extract (containing 300 ng LH). Only oxytocin increased sperm concentration of the medium (assessed by optical density measurements) compared to control incubations. Oxytocin was then tested in vivo in two groups of fish: normal males, and males that had been treated with 17alpha-methyltestosterone during larval stages to inhibit seminal vesicle development (MT males). Both groups of fish received two doses of carp pituitary suspension (8 and 10 mg/kg, respectively i.m.) with or without subsequent oxytocin treatment (5 IU/kg i.v.; cPS-OT treatment and cPS treatment, respectively). There was no effect of oxytocin on the number of strippable males. Of cPS and cPS-OT treated fish, 87% MT males and 60% normal males were strippable. The stripped semen volume was low in both groups but MT males produced higher (P < 0.001) hatching rates (63.1%) than did normal males (2.1%).
Assuntos
Peixes-Gato/fisiologia , Ocitocina/análogos & derivados , Ocitocina/farmacologia , Sêmen/fisiologia , Animais , Dinoprosta/farmacologia , Epinefrina/farmacologia , Larva/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Masculino , Metiltestosterona , Hipófise , Contagem de Espermatozoides , Testículo/efeitos dos fármacos , Testículo/fisiologia , Extratos de Tecidos/farmacologia , Vasopressinas/farmacologiaRESUMO
The effects of 17alpha-methyltestosterone on seminal vesicle development in the African catfish, Clarias gariepinus, were investigated in an attempt to improve semen collection from this species. Treatment of larvae with dietary 17alpha-methyltestosterone at 50 mg kg(-1) for days 12-33 or days 12-40 after hatching, or at 20 mg kg(-1) for days 12-26, 12-33, 12-40 or 12-47 after hatching inhibited the development of the seminal vesicle finger-like extensions in male catfish, but did not affect the sex ratio. The minimum effective dose and period of treatment to inhibit seminal vesicle development in all male catfish treated with 17alpha-methyltestosterone was 20 mg kg(-1) for days 12-40 after hatching. Male catfish from this treatment group developed normal testes that, in some cases, contained a few oocytes, which tended to disappear before sexual maturation. After sexual maturation, the semen release response was evaluated in males with incomplete seminal vesicles. Fluid with viable spermatozoa was obtained after two consecutive injections of carp pituitary suspension, from 10 of 19 males that had been fed 20 mg 17alpha-methyltestosterone kg(-1) for days 12-40 or days 12-47 after hatching, but from only 4 of 15 males that did not receive any dietary steroid. Intratesticular semen quality was not affected by 17alpha-methyltestosterone treatment. The results of this study demonstrate that the absence of seminal vesicle extensions induced by treatment with 17alpha-methyltestosterone facilitated the collection of semen by stripping from this species of fish.
Assuntos
Peixes-Gato/crescimento & desenvolvimento , Metiltestosterona/farmacologia , Sêmen , Glândulas Seminais/crescimento & desenvolvimento , Manejo de Espécimes/métodos , Análise de Variância , Animais , Distribuição de Qui-Quadrado , Depressão Química , Relação Dose-Resposta a Droga , Ejaculação/efeitos dos fármacos , Feminino , Fertilização , Masculino , Glândulas Seminais/efeitos dos fármacos , Razão de Masculinidade , Maturidade Sexual/fisiologia , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Fatores de TempoRESUMO
The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (T(chamber)) and of the semen (T(semen)) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (-2, -5, and -10 degrees C/min) and different temperatures at plunging into liquid N2. The difference between T(semen) and T(chamber) increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when T(semen) at plunging was higher than -30 degrees C to values equal to those of control when T(semen) at plunging was equal to or lower than -38 degrees C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between T(semen) and T(chamber) and improved semen survival. Semen could be plunged at temperatures as high as -36 degrees C when cooled at -5 or -10 degrees C/min, without compromising postthaw semen survival. Cooling at -2 degrees C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of -2 to -5 degrees C/min, hatching rates can be maximized by plunging as soon as T(semen) reaches -38 degrees C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of -5 to -10 degrees C/min combined with a 5-min holding period in the freezer, at -40 degrees C.
Assuntos
Peixes-Gato , Criopreservação/métodos , Preservação do Sêmen/métodos , Sêmen , África , Animais , Crioprotetores , Feminino , Fertilização , Pesqueiros , Técnicas In Vitro , Masculino , Metanol , TemperaturaRESUMO
Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.