Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?

BACKGROUND: In humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs. METHODS: Forty Nellore bulls were divided into three age groups: 1.8 to 2 years - young bulls; 3.5 to 7 years - adult bulls; and 8 to 14.3 years - aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA). RESULTS: We report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05). CONCLUSION: These results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

CONTEXTE: Chez l'homme, de nombreuses données indiquent maintenant que l'avancée de l'âge du père est associée à une réduction de l'intégrité de l'ADN des spermatozoïdes et aux échecs de développement embryonnaire. D'un autre côté, il est aussi rapporté que les jeunes pères, tels les adolescents, sont porteurs d'un risque accru d'issue défavorable de la grossesse. Ces observations suggèrent que, au moins chez l'humain, il existe une tranche d'âge dans laquelle l'intégrité de l'ADN des spermatozoïdes est optimale. Chez les bovins, on dispose de peu de connaissances sur la qualité de l'ADN des spermatozoïdes des jeunes taureaux et sur son mode d'évolution avec l'âge. La présente étude a pour but de combler ce manque car il peut être important, pour l'industrie bovine, de savoir à quelle période précise un taureau est un reproducteur optimal pour les programmes de reproduction. MATÉRIEL ET MÉTHODES: Quarante taureaux Nellore ont été répartis en trois groupes d'âge: 1, 8 à 2 ans ­ jeunes taureaux; 3,5 à 7 ans ­ taureaux adultes; et 8 à 14,3 ans ­ taureaux âgés. Trois éjaculats ont été collectés par taureau, cryopréservés et évalués pour différents paramètres incluant l'analyse assistée du sperme par ordinateur (CASA), l'intégrité des membranes plasmique et acrosomique, le potentiel mitochondrial, la protamination du noyau, l'altération oxydative de l'ADN et l'évaluation de la structure de la chromatine du noyau du spermatozoïde (SCSA). RÉSULTATS: Nous rapportons ici que les jeunes taureaux présentent des valeurs supérieures de la mobilité des spermatozoïdes et de l'intégrité des membranes plasmique et acrosomique, ainsi qu'un potentiel mitochondrial élevé. Cependant, les jeunes taureaux présentent aussi des valeurs plus élevées d'anomalies morphologiques des spermatozoïdes que celles des groupes adulte et âgé (p < 0.05). De plus, les jeunes taureaux ont une protamination plus défectueuse que celle des taureaux plus âgés. Les taureaux les plus âgés présentent plus d'altérations oxydatives du noyau que les jeunes taureaux alors que les deux groupes -jeunes et âgés- sont plus susceptibles d'avoir une dénaturation de l'ADN nucléaire comme indiqué par le SCSA (p < 0.05). CONCLUSIONS: Ces résultats indiquent que les spermatozoïdes des jeunes taureaux survivent le mieux au processus de congélation, suivis par les adultes puis les âgés. Toutefois, les jeunes taureaux et les âgés sont plus susceptibles d'avoir une altération de l'ADN, causée respectivement par une protamination déficiente et une oxydation. Par conséquent, bien que les jeunes taureaux aient des paramètres spermatiques corrects à l'évaluation classique, nos résultats indiquent que leurs spermatozoïdes peuvent présenter un certain degré d'immaturité structurale nucléaire.

Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls / Deficiências de protaminação e danos de DNA no sêmen de um touro Nelore com elevada porcentagem de patologias espermáticas em comparação a touros normospérmicos

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.(AU)

O exame de rotina de sêmen, o qual avalia a concentração de espermatozoides, a motilidade e a morfologia, pode não identificar defeitos sutis na arquitetura da cromatina de espermatozoides. Os touros parecem ter cromatina estável com baixos níveis de fragmentação do DNA. No entanto, a natureza da fragmentação e o seu impacto sobre a fertilidade ainda não estão claros e não há relatos que caracterizam a organização do DNA e os danos nessa espécie com mais detalhes. A seleção genética intensiva e o uso da inseminação artificial e da produção in vitro de embriões, além do processo de criopreservação, podem contribuir para o dano da cromatina, e sabe-se a importância da integridade do DNA espermático para o sucesso dessas tecnologias. Amostras de sêmen de três ejaculados de um touro Nelore com altos níveis de alterações morfológicas (55,8±5,1%) foram selecionadas para realização de exames complementares. Os danos de acrossoma (76,9±8,9%) e das membranas plasmáticas (75,7±9,3%), bem como quebras de fita de DNA de espermatozoides (13,8±9,5) e deficiência de protamina (3,7±0,6) foram significativamente maiores em comparação aos valores avaliados no sêmen de cinco touros Nelore com normospermia (24,3±3,3%; 24,5±6,1%; 0,6±0,5%; 0,4±0,6% para acrossoma, membrana plasmática, quebras de DNA e deficiência de protamina, respectivamente) (p<0,05). Motilidade e porcentagem de espermatozoides com baixo potencial mitocondrial não diferiram estatisticamente. Essas avaliações mostram que análises de sêmen de rotina (neste caso, morfologia) podem apontar para a extensão e a complexidade dos danos na célula espermática, o que indica que a deficiência de protamina e os danos no DNA podem ocorrer simultaneamente a defeitos morfológicos. Tal ocorrência enfatiza a importância das análises de sêmen clássicas e dos testes complementares.(AU)

Clinical aspects of antenatal glucocorticoid treatment for prevention of neonatal respiratory distress syndrome.

Main indications for antenatal administration glucocorticoid to pregnant women are premature contractions, hemorrhage during pregnancy, conditions of fetal distress and maternal diseases. There are some absolute or relative contraindications as well: severe forms of preeclampsia, diabetes mellitus, premature rupture of membranes, maternal and/intrauterine infections. In a retrospective evaluation of the data obtained at our institution of 637 nonrandomized cases from the years 1980-1985, we could demonstrate the dependence of the therapeutic results on the sex of the newborn. The RDS incidence is significantly different after betamethasone prophylaxis. It was 1/25 (4%) in girls compared to 13/31 (42%) in boys. A marked reduction of the RDS incidence is only detectable after betamethasone therapy from the 32nd to the 34th week of gestation. Thus we recommend RDS prophylaxis for all patients with premature contraction, mainly between the 32nd and 34th week of pregnancy. In addition, it should be given in cases of confirmed lung immaturity. Special restrictions are necessary in cases of preeclampsia, eclampsia, diabetes and confirmed maternal infections. In the group of diabetes or preeclampsia patients an RDS prophylaxis should only be given, if at all, when it can be performed under intensive care conditions.