Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles

Background: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval.Methods: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119 +/- 102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400 x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying >50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28 +/- 0.20 mu m, length 4.75 +/- 0.20 mu m/absence of vacuoles occupying >4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study.Results: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6 +/- 2.2% vs. 1.6 +/- 2.1% P = 0.83 and 25.2 +/- 19.2% vs. 26.1 +/- 19.0% P = 0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r = 0.57 95% CI: 0.47-0.65 P < 0.0001 and r = 0.50 95% CI: 0.38-0.58 P < 0.0001, respectively).Conclusions: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background: The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).Methods: A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 mu m and width: 3.28+/-0.20 mu m, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.Results: No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).Conclusions: The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.

Efficacy of the motile sperm organelle morphology examination (MSOME) in predicting pregnancy after intrauterine insemination

Background: Although the motile sperm organelle morphology examination (MSOME) was developed merely as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in the evaluation of semen quality. The aim of this study was to determine the prognostic value of normal sperm morphology using MSOME with regard to clinical pregnancy (CP) after intrauterine insemination (IUI).Methods: A total of 156 IUI cycles that were performed in 111 couples were prospectively analysed. Each subject received 75 IU of recombinant FSH every second day from the third day of the cycle. Beginning on the 10th day of the cycle, follicular development was monitored by vaginal ultrasound. When one or two follicles measuring at least 17 mm were observed, recombinant hCG was administered, and IUI was performed 12-14 h and 36-40 h after hCG treatment. Prior to the IUI procedure, sperm samples were analysed by MSOME at 8400x magnification using an inverted microscope that was equipped with DIC/Nomarski differential interference contrast optics. A minimum of 200 motile spermatozoa per semen sample were evaluated, and the percentage of normal spermatozoa in each sample was determined.Results: Pregnancy occurred in 34 IUI cycles (CP rate per cycle: 21.8%, per patient: 30.6%). Based on the MSOME criteria, a significantly higher percentage of normal spermatozoa was found in the group of men in which the IUI cycles resulted in pregnancy (2.6+/-3.1%) compared to the group that did not achieve pregnancy (1.2+/-1.7%; P = 0.019). Logistic regression showed that the percentage of normal cells in the MSOME was a determining factor for the likelihood of clinical pregnancy (OR: 1.28; 95% CI: 1.08 to 1.51; P = 0.003). The ROC curve revealed an area under the curve of 0.63 and an optimum cut-off point of 2% of normal sperm morphology. At this cut-off threshold, using the percentage of normal sperm morphology by MSOME to predict pregnancy was 50% sensitive with a 40% positive predictive value and 79% specificity with an 85% negative predictive value. The efficacy of using the percentage of normal sperm morphology by MSOME in predicting pregnancy was 65%.Conclusions: The present findings support the use of high-magnification microscopy both for selecting spermatozoa and as a routine method for analysing semen before performing IUI.

The effects of male age on sperm analysis by motile sperm organelle morphology examination (MSOME)

Background: This study aimed to investigate the influence of age on sperm quality, as analysed by motile sperm organelle morphology examination (MSOME).Methods: Semen samples were collected from 975 men undergoing evaluation or treatment for infertility. Sperm cells were evaluated at 8400x magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. Two forms of spermatozoa were considered: normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV, defined as vacuoles occupying > 50% of the sperm nuclear area). At least 200 spermatozoa per sample were evaluated, and the percentages of normal and LNV spermatozoa were determined. The subjects were divided into three groups according to age: Group I, less than or equal to 35 years; Group II, 36-40 years; and Group III, greater than or equal to 41 years.Results: There was no difference in the percentages of normal sperm between the two younger (I and II) groups (P > 0.05). The percentage of normal sperm in the older group (III) was significantly lower than that in the younger (I and II) groups (P < 0.05). There was no difference in the percentage of LNV spermatozoa between the younger (I and II) groups (P > 0.05). The percentage of LNV spermatozoa was significantly higher in the older group (III) than in the younger (I and II) groups (P < 0.05). Regression analysis demonstrated a significant decrease in the incidence of normal sperm with increasing age (P < 0.05; r = -0.10). However, there was a significant positive correlation between the percentage of spermatozoa with LNV and male age (P < 0.05, r = 0.10).Conclusion: The results demonstrated a consistent decline in semen quality, as reflected by morphological evaluation by MSOME, with increased age. Considering the relationship between nuclear vacuoles and DNA damage, these age-related changes predict that increased paternal age should be associated with unsuccessful or abnormal pregnancy as a consequence of fertilisation with damaged spermatozoa. Given that sperm nuclear vacuoles can be evaluated more precisely at high magnification, these results support the routine use of MSOME for ICSI as a criterion for semen analysis.

GnRH agonist versus GnRH antagonist in assisted reproduction cycles: oocyte morphology

Background: The selection of developmentally competent human gametes may increase the efficiency of assisted reproduction. Spermatozoa and oocytes are usually assessed according to morphological criteria. Oocyte morphology can be affected by the age, genetic characteristics, and factors related to controlled ovarian stimulation. However, there is a lack of evidence in the literature concerning the effect of gonadotropin-releasing hormone (GnRH) analogues, either agonists or antagonists, on oocyte morphology. The aim of this randomized study was to investigate whether the prevalence of oocyte dysmorphism is influenced by the type of pituitary suppression used in ovarian stimulation.Methods: A total of 64 patients in the first intracytoplasmic sperm injection (ICSI) cycle were prospectively randomized to receive treatment with either a GnRH agonist with a long-term protocol (n: 32) or a GnRH antagonist with a multi-dose protocol (n: 32). Before being subjected to ICSI, the oocytes at metaphase II from both groups were morphologically analyzed under an inverted light microscope at 400x magnification. The oocytes were classified as follows: normal or with cytoplasmic dysmorphism, extracytoplasmic dysmorphism, or both. The number of dysmorphic oocytes per total number of oocytes was analyzed.Results: Out of a total of 681 oocytes, 189 (27.8 %) were morphologically normal, 220 (32.3 %) showed cytoplasmic dysmorphism, 124 (18.2%) showed extracytoplasmic alterations, and 148 (21.7%) exhibited both types of dysmorphism. No significant difference in oocyte dysmorphism was observed between the agonist- and antagonist- treated groups (P > 0.05). Analysis for each dysmorphism revealed that the most common conditions were alterations in polar body shape (31.3%) and the presence of diffuse cytoplasmic granulations (22.8%), refractile bodies (18.5%) and central cytoplasmic granulations (13.6%). There was no significant difference among individual oocyte dysmorphisms in the agonist- and antagonist-treated groups (P > 0.05).Conclusions: Our randomized data indicate that in terms of the quality of oocyte morphology, there is no difference between the antagonist multi-dose protocol and the long-term agonist protocol. If a GnRH analogue used for pituitary suppression in IVF cycles influences the prevalence of oocyte dysmorphisms, there does not appear to be a difference between the use of an agonist as opposed to an antagonist.

Lung morphology and growth of rats exposed to tobacco smoke and alcohol

OBJETIVO: Investigar os efeitos morfológicos da exposição crônica à inalação de fumaça do tabaco e o do consumo de álcool nos pulmões e no crescimento de ratos. MÉTODOS: Sessenta ratos Wistar machos foram distribuídos em quatro grupos: controle, tabaco, álcool e tabaco + álcool, e acompanhados por um período de 260 dias. No final do periodo foi realizada análise morfológica dos pulmões por microscopia óptica e eletrônica. O crescimento dos ratos foi investigado através da medição do comprimento focinho-ânus, peso corporal e índice de massa corporal. RESULTADOS: Os três grupos expostos às drogas apresentaram peso e comprimento significativamente menores que os do grupo controle. As percentagens de bronquiolite e alveolite, e o diâmetro alveolar médio foram maiores nos grupos expostos à fumaça do tabaco, mas sem significancia estatística quando comparadas ao grupo controle. A microscopia eletrônica revelou apoptose mais intensa e lesões degenerativas no grupo de fumantes, enquanto lesões degenerativas nos corpos lamelares foram mais intensas com a associação de ambas as drogas. CONCLUSÕES: Este modelo experimental mostrou alterações morfológicas observadas por microscopia eletrônica, principalmente devido à exposição ao tabaco. Tanto o alcool como o tabaco prejudicaram o crescimento dos animais, o tabaco mostrando um efeito maior sobre o comprimento e o álcool sobre o peso corporal.

Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus)

This study describes the influence of incubation temperature during initial development phase on the morphology and muscle growth characteristics in the pacu (Piaractus mesopotamicus). Pacu eggs were incubated at 25, 27, and 29 degreesC until hatching. After day 5, fish from each temperature were transferred to 5001 tanks. At hatching and after 5, 25, and 60 days, muscle samples were collected, some were frozen in liquid nitrogen and others fixed in 4% paraformaldehyde or 2.5% glutaraldehyde. These samples were used for morphological, histochemical, immunohistochemical, and morphometric analysis. At hatching, we observed a superficial monolayer of small diameter fibers, lying just beneath the skin surrounding several round cells. From day 5, we observed two distinct populations of muscle fibers distributed in two layers: (1) red-in a superficial region with aerobic activity, and following acid preincubation, high mATPase activity, and 2) white-with anaerobic activity, and following alkaline preincubation, high mATPase activity. Twenty-five days after hatching, an intermediate layer and cell proliferating zones could be seen in the dorsal fin muscle region, with intermediate characteristics. Throughout the experimental period, there was an increase in muscle mass due to new fiber recruitment in the cell proliferating zones and between the more differentiated fibers in red, intermediate, and white muscles. This was more obvious from day 25, and at 29 degreesC than at 25 and 27 degreesC. Fiber hypertrophy occurred from hatching to 60 days and was more evident from 5 to 25 days. The number of proliferating nuclei (PCNA-labelling) increased from hatching to 60 days, and was more obvious in the 29 degreesC group at 60 days. Our results show that at incubation temperatures of 25, 27 and 29 degreesC, hypertrophy was predominantly from hatching to 25 days, after that muscle growth by hyperplastic mechanism increased. The interaction of muscle hypertrophic and hyperplastic growth processes in the 29 degreesC group produced the largest fish at the end of the experiment. (C) 2004 Elsevier B.V. All rights reserved.

Alternative calcium source effects on commercial egg production and quality

This study aimed at verifying the possibility of replacing calcitic limestone by marine calcium in the diet of layers. A total number of 321 Hi-sex hens, with 40 weeks of age at the beginning of the experiment, was used. A completely randomized experimental design was applied, with 5 treatments (0, 15, 30, 45, and 60 % of calcitic limestone replacement by marine calcium source) and eight replicates of eight birds each. Treatments significantly affected specific gravity (p<0.05), with the inclusion of 60% marine calcium (T5) presenting the worst result as compared to T1, which included only calcitic limestone as calcium source. It was concluded that marine calcium can replace up to 45% of calcitic limestone with no effects on performance or egg quality.

Beak trimming methods and their effect on the performance and egg quality of Japanese quails (Coturnix japonica) during lay

A total number of 720 Japanese quail females, reared under identical management conditions, and receiving feed and water ad libitum, was used. Quails were housed and reared from 1-35 days of age in litter pens in a rearing house, and then were transferred to a quail layer house. This study aimed at verifying if different beak trimming method could optimize egg production and quality by reducing stress and cannibalism among birds. A completely randomized experimental design was used, with six treatments in a 2x3 factorial arrangement, with two beak trimming ages (14 and 21 days) and three beak trimming variants (non-trimmed, 1/3 of the beak trimmed, or 1/2 of the beak trimmed), 6 replicates of 20 birds each. Performance was evaluated by feed intake, percentage of lay, egg mass, feed conversion ratio per dozen and per kg eggs, and mortality. Performance was influenced by trimming variant, with the best results obtained by birds with intact and those with 1/3 of the beak trimmed. Better specific gravity was in quails submitted to beak trimming at 21 days of age, whereas other egg quality parameters did not present significant results. Based the results obtained in the present study, it is recommended to submit to beak trimming either at 14 or 21 days of age.

Effect of organic mineral supplementation on the egg quality of semi-heavy layers in their second cycle of lay

This study was carried out to evaluate the effects of dietary trace mineral levels and sources on egg quality parameters of second-cycle semi-heavy layers. A number of 360 72-week-old layers were submitted to forced molting. Upon return of lay (83 weeks of age), birds were distributed according to a completely randomized experimental design of six treatments with six replicates of 10 birds each. The control treatment consisted of 0.10% dietary supplementation of trace minerals from inorganic sources, which was proportionally replaced by five levels (110, 100, 90, 80, 70%) of an organic trace mineral supplement containing 30, 30, 40, 6, 0.61, and 0.3 g/kg product of Zn, Fe, Mn, Cu, I, and Se, respectively. All diets contained equal protein, energy, and amino acid levels. Every 28 days of the experimental period (112 days) four eggs per replicate were collected for egg quality evaluation. The following parameters were evaluated: specific gravity, yolk, albumen and eggshell percentages, yolk index, Haugh units, and eggshell thickness and breaking strength. One sample per replicate, consisting of the pool of the yolks of three eggs collected at the end of each experimental period, was used to assess protein and mineral (Ca, P, Cu, Fe, Mn, and Zn) contents. The results were submitted to ANOVA, and means to the test of Tukey at 5% significance level. The evaluated trace mineral levels and sources did not influence any of the studied egg quality parameters. It was concluded that reducing organic trace mineral supplementation in up to 70% relative to 100% inorganic trace mineral supplementation does not affect egg parameters and therefore, can be applied to the diet of semi-heavy layers in their second cycle of lay.

Production performance and egg quality of quails (Coturnix japonica) during several periods of the day

Three experiments were carried out to analyze the performance and egg quality of Japanese quails throughout the day when submitted to different lighting programs. In each experiment, birds were submitted to a period of 28 days for adaptation to the lighting program. During the following three days, each day - from 7 am to 7 pm or 9 pm - was divided into six or seven periods of two hours each, and the remaining hours corresponded to another single period. All birds were submitted to the same management practices, and received water and feed ad libitum. The experimental diet was formulated according to NRC (1994) standards. It was observed lower feed intake in the period of 9 pm to 7 am, and a higher incidence of lay from 3 pm to 7 pm, as opposite to hens, which peak of egg laying occurs during the morning. Some controversial results were found among experiments as to eggshell quality during the different periods of the day. Variation on the lighting program had little influence on the other performance and egg quality parameters.

Effect of the calcium level and limestone particle size on the performance of semi-heavy layers in the second cycle of egg production

An experiment was carried out at the Research and Development Unit of Brotas aiming at evaluating dietary calcium level and limestone particle size on the production performance of commercial (Hy-Line Brown) layers in the second lay cycle. Experiment duration was 112 days. A total number of 288 hens, with 83 weeks of age in the beginning of the experiment, were used in a completely randomized experimental design in a factorial arrangement of 2x3, with two calcium levels (3.5 and 4.0%) and three limestone particle size compositions: 100% fine limestone (FL), 30% coarse limestone (CL) + 70% fine limestone (FL), and 50% (CL) + 50% (FL), with six replicates of eight birds each. Egg weight (g), egg production (%), egg mass (%), feed intake (g), feed conversion ratio (kg/dz and kg/kg), mortality (%), and egg loss (%) were evaluated. The analysis of variance did not detect significant differences (p>0.05) among treatments on any of the evaluated performance parameters. It was concluded that the tested calcium levels and limestone particle composition did not influence the performance of semi-heavy layers in second production cycle.

Forced-Molting methods and their effects on the performance and egg quality of japanese quails (Coturnix japonica) in the second laying cycle

The experiment was carried out in the experimental poultry house of the Research and Development Unit of Brotas of Agência Paulista de Tecnologia dos Agronegócios do Centro-Oeste, SP, Brazil. The objective of the study was to evaluate the performance of Japanese quails submitted to forced molting aiming at optimizing the use of the same quail flock by promoting a second laying cycle. A total number of 400 67-day-old Japanese quails in lay, previously submitted to 14 days of forced molting, was distributed in a completely randomized experimental design into five treatments (T1= not submitted to forced molting, T2= 03 days of fasting + fed ad libitum, T3= 01 days of fasting + 13 days of feed restriction, T4= 02 days of fasting + 12 days of feed restriction, and T5= 03 days of fasting + 11 days of feed restriction. Feeds were contained equal nutrient levels, and were formulated according to NRC (1994) recommendations. There were significant differences among the studied treatments. Although the treatment of 3 days of fasting followed by ad libitum feeding resulted in lower egg weight, it promoted better lay percentage, egg mass, and feed conversion ratios (FCR/dz and FCR/kg). on the other hand, 3 days of fasting followed by restricted feeding resulted in higher feed intake and worse feed conversion ratios (FCR/dz and FCR/kg). When birds were not submitted to forced molting, they presented lower lay percentage and egg mass.