Comparison of apoptotic cells between cryopreserved ejaculated sperm and epididymal sperm in stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5 C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5 C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computerassisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.

Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells

Objective: To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH).Study design: Patients without ovulatory dysfunction, aged <= 37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test.Results: DNA fragmentation: 32 patients were included in either the GnRH agonist group (n = 16) or the antagonist group (n = 16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P = 0.76) between the agonist group (15.5 +/- 9.4%) and the antagonist group (18.8 +/- 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n = 14) or the antagonist group (n = 14). The percentage of GCs positive for apoptosis did not differ significantly (P = 0.78) between the agonist group (34.6 +/- 14.7%) and the antagonist group (36.5 +/- 22%).Conclusions: The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. (C) 2012 Elsevier B.V. All rights reserved.

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.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cryopreservation and fertility of ejaculated and epididymal stallion sperm

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological aspects of spermatogenic cells at different stages of sexual maturation in black isogenic C57BL/6/Uni mice

In order to study the morphological changes that occur in cells of the testes of isogenic black mouse C57BL/6/Uni into three periods during spermatogenetic used 15 mice divided into 3 groups of 5 animals with 40,50 and 60 days of age. The mice were sacrificed and weighed. Testicles were weighed and measured, and histologically processed and stained with HE, PAS and Masson Massom-H and evaluated under light microscopy. It was observed that group I with 40 days of age in the seminifcrous tubules had a lumen with sparse small amount of interstitial tubular cells. In the seminiferous epithelium type A spermatogonia, intermediate and B were identified, which occupied the compartment adbasal and intermingled with these cells in spermatocytes I in Pachytene and leptotene was observed, whereas in the adluminal compartment Golgi phase spermatids we observed the presence of acrosomal granule. In group II, the cells of the seminiferous epithelium were developed and it was observed in round spermatids cephalic hood phase plus many elongated spermatids in acrosome phase and Sertoli cells. In Group III, 60 days old, it was found that seminiferous epithelium which was of the tubules had elongated spermatids in acrosome phase and maturation, with elongated nuclei and acrosomal system typical of spermiation in the presence of sperm and residual bodies near the tubular lumen. Therefore morphological evolution of germ cell testicular spermatids can be checked and recognized in its four phases: Golgi, cap, acrosome and maturation over the age of the animal.

Ultrastructure of spermatogenic cells and spermatozoa in Hoplias malabaricus (teleostei, characiformes, erythrinidae)

The differentiation of spermatids in Hoplias malabaricus is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation, and excess cytoplasm elimination. In the resulting spermatozoon, the head is round and the nucleus contains chromatin compacted in thick filaments, peripherically arranged, to a central electron-lucent area. The acrosome is absent. The nuclear fossa is eccentric but not pronounced. The proximal centriole penetrates it and is oblique to the flagellum. The long midpiece has several converging elongate vesicles, forming membranous hoops in the initial segment of the flagellum, but has no cytoplasmic channel. The mitochondria are elongate and branched or C-shaped and located around the initial segment of the axoneme. The lateral flagellum does not show lateral projections. The ultrastructural characteristics of H. malabaricus spermatozoa are similar to the Cypriniformes. (C) 2001 the Fisheries Society of the British Isles.

Persistent Impairment of Testicular Histology and Sperm Motility in Adult Rats Treated with Cisplatin at Peri-Puberty

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Orchidopexy restores morphometric-stereologic changes in the caput epididymis and daily sperm production in cryptorchidic mice, although sperm transit time and fertility parameters remain impaired

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphofunctional changes in Leydig cells throughout the continuous spermatogenesis of the freshwater teleost fish, Serrasalmus spilopleura (Characiformes, Characidae): an ultrastructural and enzyme study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Daily sperm production and testicular morphometry in goats according to external scrotal conformation

The present study aimed to compare testicular histology and the testicular cell population as well as spermatogenic efficiency in goats with different scrotal conformations. Eighteen goats were divided into 3 groups: Group I - goats without bipartition of the scrotum, Group II - animals with bipartition of the scrotum up to 50% of the testicular length, Group III - goats with scrotal bipartition more than 50% of the testicular length. In goats in Groups I, II and III, the values for the volume density of seminiferous epithelium were 68.9 +/- 0.6%, 71.5 +/- 2.8% and 73.4 +/- 4.7% (P < 0.05), the height of the seminiferous epithelium were 60.2 +/- 4.9 mu m, 61.0 +/- 5.0 mu m and 73.1 +/- 6.6 mu m (P < 0.05), total length of seminiferous tubules found for Groups I, II and III were 2091.9 +/- 27 m, 2172.5 +/- 24.1 m, and 2340.1 14 m (P < 0.05), number of Sertoli and Leydig cells were 1.8 +/- 0.4 x 10(9) and 1.4 +/- 0.1 x 10(9), 2.2 +/- 0.4 and 2.2 +/- 0.7 x 10(9), and 2.5 +/- 0.1 10(9) and 2.3 +/- 0.510(9)(P < 0.05)and daily sperm production observed were 2.1 0.3 x 109, 2.8 0.4 x 109, and 3.1 0.7 x 109 (P < 0.05). In conclusion, goats with greater scrotal bipartition have a greater capacity to produce reproductive cells that is reflected in a greater reproductive potential. (C) 2011 Elsevier B.V. All rights reserved.

The effects of male age on sperm DNA damage in an infertile population

The objective was to investigate the influence of age on sperm DNA damage. Semen samples were collected from 508 men in an unselected group of couples attending infertility investigation and treatment. DNA fragmentation in spermatozoa was measured by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay; at least 200 spermatozoa in randomly selected areas of microscope slides were evaluated using a fluorescent microscope and the percentage of TUNEL positive spermatozoa was determined. The number of cells with red fluorescence (TUNEL positive) was expressed as a percentage of the total sample [DNA fragmentation index (DFI)]. Age was treated as a continuous variable for regression and correlation analysis. The following male age groups were used: Group I: ≤35 years, Group II: 36-39 years, and Group III: ≥40 years. DFI was significantly lower in Group I than in Group II (P = 0.034) or III (P = 0.022). There was no difference in DFI between Groups II and III. In addition, regression analysis demonstrated a significant increase in sperm DFI with age (P = 0.02). TUNEL assay clearly demonstrates an increase in sperm DNA damage with age. © 2007 Published by Reproductive Healthcare Ltd.