RESUMO
Sperm deep freezing procedures for ram semen have considerable variations regarding the steps being employed for cooling, freezing, and addition of cryoprotectants. In this work, we evaluated the effects of the addition of glycerol and/or the disaccharides sucrose and trehalose to hypertonic diluents either before or after cooling from 30 °C to 5 °C in Merino Australian ram semen cryopreservation. Using optical and transmission electron microscopy techniques, we assessed that glycerol was beneficial to the cooling process independently of its addition at 30 °C or 5 °C in terms of sperm membrane integrity in different regions of the plasma membrane (acrosomal region, 14.5% higher integrity; postacrosomal region, 8.0% higher integrity [P < 0.01]; hypoosmotic swelling test [HOST], 10.8% higher integrity [P < 0.001]). Disaccharides were necessary for a better cryopreservation in liquid nitrogen, and the best procedure was their addition after cooling at 5 °C (12% higher sperm motility [P < 0.001]; 8% higher acrosome integrity, [P < 0.05]; 9.5% higher plasma membrane integrity assessed by HOST [P < 0.001]). Trehalose showed a greater preservation cryoprotectant capacity than sucrose, as indicated by sperm motility after thawing (8.1% greater [P < 0.01]) and by the integrity of the intermediate piece (20% greater [P < 0.05]). From these results, we conclude that the best procedure for ram semen cryopreservation in hypertonic disaccharide-containing diluents is the addition of glycerol and trehalose after the cooling process, at 5 °C.
Assuntos
Criopreservação/veterinária , Glicerol/farmacologia , Preservação do Sêmen/veterinária , Ovinos/fisiologia , Sacarose/farmacologia , Trealose/farmacologia , Animais , Criopreservação/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Congelamento , Glicerol/química , Masculino , Preservação do Sêmen/métodos , Sacarose/química , Trealose/químicaRESUMO
In Northern Patagonia, Argentina, the ovine mating season starts on March 15, which is the time when rams are submitted to summer temperatures. This study assessed the adaptability of 12 Australian Merino rams, six unshorn and six shorn, half of which were treated in a heat chamber for five days (09.00 hours to 17.00 hours) that gradually reached 40 °C. In an attempt to quantify the effects of heat stress on sperm head morphology, ellipticity was analyzed to establish the relationship between the distributions of subpopulations, light hours, temperature and humidity. Ellipticity was measured on 9224 sperm heads that were obtained over 12 weeks starting in the summer time. Four sperm head subpopulations (S) were identified by comparison with a sperm head population of ejaculates obtained in the late breeding season without the effect of heat stress (S1 = heads with ellipticity ≥ 2.00; S2 = sperm head with range of ellipticity between 1.80 and 1.99; S3 = sperm head with range of ellipticity from 1.60 to 1.79; and S4 = sperm head with range of ellipticity from 1.30 to 1.59). The variable sperm head ellipticity for each ejaculate was expressed as the means and frequencies of subpopulation. The results demonstrate changes in ram sperm head ellipticity in different conditions (control/treated, unshorn/shorn) throughout the experiment (P < 0.05). Treated shorn rams had a higher mean ellipticity and frequency of elliptical heads (mean ellipticity value = 2.06 and S1 frequency = 76.35%), peaking in the seventh week posttreatment (on the basis of the action of heat stress on seminiferous tubules). According to this study, unshorn rams were better adapted to heat stress than the shorn ones.
Assuntos
Ovinos/fisiologia , Espermatozoides/citologia , Estresse Fisiológico/fisiologia , Animais , Argentina , Masculino , Análise do Sêmen/veterinária , Espermatozoides/fisiologiaRESUMO
Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.
Assuntos
Membrana Celular/ultraestrutura , Criopreservação , Cabeça do Espermatozoide/ultraestrutura , Animais , Membrana Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Congelamento , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Eletrônica de Transmissão , Carneiro Doméstico , Cabeça do Espermatozoide/efeitos dos fármacosRESUMO
We evaluated freeze-thawing tolerance of heterospermic ram spermatozoa (Pampinta breed) in a base diluent (Tris, citric acid, fructose, egg yolk, glycerol) with the addition of different trehalose concentrations (0-400 mOsm). We chose sperm motility, acrosome integrity and hypo-osmotic swelling test as parameters to evaluate cryopreservation capacity. We obtained the best results for 50 and 100 mOsm trehalose-supplemented extenders, with values (referred to fresh semen values) of 65% for motility, 75% for acrosome integrity and 50% for hypo-osmotic swelling test, while freeze-thawing tolerance diminished significantly for 200 and 400 mOsm of the disaccharide. Fertility values measured at lambing were 47.1 and 44.6% (2 consecutive years), using semen cryopreserved in 100 mOsm trehalose-containing diluent, which is 2.5 times greater than those obtained with the base diluent (18.5 and 14.5%). We conclude that the membrane-protecting disaccharide trehalose confers a greater cryoprotective capacity to the base extender, when added up to 100 mOsm. This action is reflected in the different sperm membranes, the motile activity and in vivo fertility.
Assuntos
Criopreservação/veterinária , Fertilidade , Preservação do Sêmen/veterinária , Ovinos , Trealose/administração & dosagem , Acrossomo/ultraestrutura , Animais , Tamanho Celular , Citratos , Feminino , Temperatura Alta , Soluções Hipotônicas , Inseminação Artificial/veterinária , Masculino , Concentração Osmolar , Preservação do Sêmen/métodos , Citrato de Sódio , Motilidade dos Espermatozoides , Espermatozoides/ultraestruturaRESUMO
To obtain better ram semen extenders for artificial insemination (AI), we developed effective trehalose-containing hypertonic diluents. The cryoprotective action of trehalose has been explained by its dehydrating activity and interaction with cell membranes. Accordingly, we tested the cryopreserving capacity of different combinations of a Salamon's modified plus trehalose extender with EDTA. Evaluations were based on the percentage of motile spermatozoa and acrosome integrity, measured after thawing and after a 4-h post-thaw resistance test at 37 degrees C. We conclude that the combination of trehalose plus EDTA confers the highest cryopreserving activity tested, not only for freeze-thawing but also for post-thawing resistance, possibly by removing calcium from the medium thereby preventing cation competition with trehalose for membrane-binding sites.