RESUMO
Germplasm preservation is crucial for reproductive programs involving farm and endangered species. This study describes the effects of slow-uncontrolled cryopreservation protocols on bovine sperm associated with testicular or epididymal tissues. Samples from the testis or epididymis (cauda) were cut into â¼0.5 or 1 cm3 fragments and cryopreserved using Me2SO (Dimethyl Sulfoxide) or glycerol-based cryoprotectants. Sperm were collected from testicular or epididymal tissue before and after freezing-thawing (38 °C or 40 °C) and kept at room temperature (RT) or 4 °C during handling. The parameters studied were viability, membrane integrity (HOS), motility, acrosome integrity, chromatin, and morphology. Pre-freezing parameters were lower in testicular sperm than epididymal: HOS+ and DNA integrity (P < 0.05). Normal-% pre-freezing testicular sperm morphology was lower than epididymal (43.3 ± 1.8% vs. 65.3 ± 14.8%). All testicular RT-kept sperm parameters decreased post-freezing, except for acrosome integrity, which remained constant (P > 0.05). There were no differences in Me2SO-frozen tissue sizes (P > 0.05). All epididymal RT-kept sperm parameters dropped post-freezing except for the constant DNA integrity (P > 0.05). 4oC-kept sperm were fitter than those at RT (P < 0.05). 4oC-kept testicular sperm viability, DNA, and membrane integrities declined after 38 °C or 40 °C thawing (P < 0.05). Acrosome integrity and motility remained unchanged after freezing (P > 0.05). 4oC-kept epididymal sperm acrosome integrity, motility, and HOS+% severely dropped post-thawing (P < 0.05). Viability and DNA integrity were unchanged (38 °C vs. 40 °C; P > 0.05). Overall, post-freezing sperm morphology was unaffected (P > 0.05), but Dag defect was significantly lower in testicular samples (P < 0.05). Whole-epididymis parameters were maintained up to 24h at 4 °C (P > 0.05). In conclusion, testis-epididymis freezing protocols should use small tissue pieces, Me2SO-based cryoprotectants, and 4°C-kept samples to reduce sperm damage.
Assuntos
Criopreservação , Preservação do Sêmen , Animais , Bovinos , Masculino , Congelamento , Criopreservação/métodos , Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Crioprotetores/farmacologia , DNA , Preservação do Sêmen/métodosRESUMO
Effective tools for male contraception are important in the control of reproduction in animal populations. The aim of the present study was to evaluate the effects of active immunization against gonadotropin-releasing hormone (GnRH) on male reproductive function assessing testicular morphological changes and serum-gonadotropin levels in pre-pubertal rabbits, guinea pigs and ram lambs. An anti-GnRH vaccine was developed by linking a GnRH-homologous molecule to a tetanus clostridial toxoid (Al(OH)3 coadjuvant). After vaccination protocols testicular morphometry, histopathological alterations and endocrine responses (FSH, LH, testosterone and cortisol serum levels) were evaluated. Testicular volume was significantly reduced in vaccinated animals with respect to the control group in rabbits, guinea pigs and ram lambs (P<0.05 to P<0.001). The anti-GnRH vaccine generated a reduction in testicular volume of 15-, 27- and 11-fold, respectively. Tubule diameters decreased in the vaccinated group with respect to the control ~2.0-, 1.2- and 3.5-fold, respectively (P<0.001). Tubule, intertubular and lumen volumes significantly decreased in vaccinated rabbits (P<0.05), guinea pigs and ram lambs (P<0.01). Vaccinated animals of the three species showed significant reductions in spermatogonial numbers (10- to 40-fold; P<0.01). Sperm was absent in all seminiferous tubules of all rabbits, and most individuals of guinea pigs (80%) and ram lambs (60%). No significant differences were observed between vaccinated and control groups regarding FSH and LH during the experiments in the three experimental species/models used. Testosterone, however, was only significantly lower (~22-fold, P<0.01) in vaccinated rabbits. In conclusion, the present study demonstrated that pre-pubertal active immunization against GnRH leads to endocrine disruption and marked differences on testicular morphometry, development and activity among lagomorphs, hystricomorphs and ovine species with species-specific sensitivity regarding the anti-GnRH immune response.
Assuntos
Hormônio Liberador de Gonadotropina/imunologia , Testículo/crescimento & desenvolvimento , Testosterona/sangue , Vacinas Anticoncepcionais/imunologia , Animais , Animais Domésticos , Pesos e Medidas Corporais , Cobaias , Imunização/veterinária , Masculino , Coelhos , Ovinos , VacinaçãoRESUMO
Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode's complete medium with heparin (TCMH; a capacitating medium containing Ca2+, NaHCO3 and heparin), Tyrode's complete medium heparin-free (TCM; a medium containing just Ca2+ and NaHCO3) or Tyrode's basal medium (TBM; a non-capacitating medium free of Ca2+, NaHCO3 and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=-0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.
Assuntos
Bovinos/fisiologia , Capacitação Espermática , Espermatozoides/citologia , Espermatozoides/fisiologia , Animais , MasculinoRESUMO
The aim of this study was to determine whether computerised sperm head morphometric analysis can be used as a diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa. Ejaculates from five bulls (4 ejaculates/bull) were pooled and processed for computerised morphometric analysis, and SYBR-14 green/ethidium homodimer-1 fluorescence-based live/dead viability assay was used simultaneously to confirm the viability index of frozen-thawed spermatozoa. Sperm samples were assigned to three experimental groups. The first group was enriched in live spermatozoa (after a double Percoll selection), the second group was enriched in dead spermatozoa (after a refreeze-thaw procedure), and the last group was a 50 : 50 pool of live/dead spermatozoa (from first and second group samples). There were significant differences (P < 0.001) related to sperm morphometric dimensional parameters among the three groups analysed, being the lowest overall sperm head dimension found in the second (dead spermatozoa) group. In conclusion, sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa.
Assuntos
Criopreservação , Espermatozoides/citologia , Animais , Fenômenos Biofísicos , Bovinos , MasculinoRESUMO
The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.
Assuntos
Alouatta/anatomia & histologia , Animais de Zoológico/anatomia & histologia , Espermatozoides/citologia , Bem-Estar do Animal , Animais , Processamento de Imagem Assistida por Computador , Incidência , Masculino , Espermatozoides/classificaçãoRESUMO
The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldi's monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods. Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (µm(2))], perimeter [P (µm)], length [L (µm)] and width [W (µm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L-W)/(L+W)] and rugosity [(4πA/P(2))]). Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56µm(2), W=2.75±0.42µm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92µm(2), L=5.06±0.49µm, W=3.51±0.31µm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75µm(2), W=3.47±0.29µm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38µm(2), L=4.90±0.57µm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72µm(2) and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62µm(2) and elongation=0.19±0.04). There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed. In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates. Broadening our knowledge of C. goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species.
Assuntos
Callimico/anatomia & histologia , Cabeça do Espermatozoide/ultraestrutura , Animais , Conservação dos Recursos Naturais , Processamento de Imagem Assistida por Computador , Masculino , Microscopia de Contraste de Fase/veterinária , Análise de Componente PrincipalRESUMO
In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.