Morfologia espermática em touros: quais os principais defeitos e o porquê ocorrem / Sperm morphology in bulls: which are the main defects and why they occur

A morfologia espermática é uma característica seminal que está associada à fertilidade e é, portanto, um componente importante na rotina do controle de qualidade em Centros de Coleta e Processamento de Sêmen (CCPS). É utilizada para caracterizar e quantificar os vários tipos de anormalidades nas células espermáticas do touro, usando preparação em montagem úmida para microscopia de Contraste de Interferência Diferencial (DIC), Contraste de Fase (CF) e Lâmina Corada (LC). O perfil da morfologia espermática permite a tomada de decisão sobre o aproveitamento ou descarte de um ejaculado para criopreservação e predizer a fertilidade de uma amostra de sêmen. Alterações no espermograma, com evidências do histórico e exame físico do touro, levam a interpretação da função testicular anormal, e consequentemente, ao diagnóstico, tratamento e prognóstico, se for o caso. As causas mais comuns de uma espermatogênese anormal nos touros incluem: termorregulação testicular anormal, desequilíbrios hormonais, estresse de manejo, ambiental e nutricional, efeitos de toxinas ou expressão de genes deletérios, além de defeitos de origem genética. Conhecendo-se a estrutura do espermatozoide, torna-se mais fácil a identificação das anormalidades espermáticas bem como a descrição das mesmas e uma melhor compreensão de sua origem.(AU)

Sperm morphology is a seminal trait that is associated with fertility and is therefore an important component of routine quality control in Semen Collection and Processing Centers (SCPC). It is used to characterize and quantify the various types of abnormalities in bull sperm cells, using wet mount preparation for Differential Interference Contrast (DIC), Phase Contrast (PC) and Slides Smear (SS) microscopy. The sperm morphology profile allows decision making on the use or disposal of an ejaculate for cryopreservation and predicts the fertility of a semen sample. Changes in sperm morphology may indicate that fertility may be impacted and therefore it is important to routinely monitor ejaculates. An abnormal spermogram with evidence from a history and physical examination of the bull can lead to the interpretation of abnormal testicular function, and consequently, to the diagnosis, treatment and prognosis, if any. The most common causes of abnormal spermatogenesis in bulls include: abnormal testicular thermoregulation, hormonal imbalances, management, environmental and nutritional stress, effects of toxins or expression of deleterious genes, in addition to defects of genetic origin. Knowledge in the structure of the sperm makes easier the identification of sperm abnormalities, as well as their description and provides a better understanding of their origin.(AU)

Geotrichum candidum as a possible cause of bovine abortion.

Geotrichum spp. are ubiquitous, saprotrophic fungi found in soil, organic matter, and silage, as a contaminant in food products and in the digestive tracts of mammals. The current study reports a case of Geotrichum candidum infection with dermatitis in an aborted bovine fetus with skin and lung lesions. A 6-month-old aborted male Holstein Friesian fetus displayed unusual lesions on the skin of the abdomen, thorax, and head, which was excessively thickened and wrinkled. These changes corresponded to orthokeratotic hyperkeratosis, neutrophil accumulation in the stratum corneum, a pyogranulomatous inflammatory infiltrate, and superficial dermal necrosis. Moderate suppurative multifocal pneumonia was observed. Large numbers of mononuclear cells and occasional fibrin thrombi within blood vessels were found in the lungs, brain, and cerebellum. Gridley staining revealed fungal structures within the skin lesions. The mycological exam demonstrated the growth of G. candidum, and phase contrast microscopy conducted on the abomasal fluid revealed hyphae compatible with this agent. The skin lesions observed, in association with the fungus isolated, indicated that the abortion was due to G. candidum infection of the bovine fetus.

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii).

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.

Four new species of Ligophorus (Monogenea: Dactylogyridae) parasitic on Mugil liza (Actinopterygii: Mugilidae) from Guandu River, southeastern Brazil.

Four species of Ligophorus (Monogenea: Dactylogyridae), i.e., L. tainhae n. sp., L. brasiliensis n. sp., L. guanduensis n. sp., and L. lizae n. sp., are described. The specimens were collected from the gills of Mugil liza (Mugilidae) from the Guandu River (22 degrees 48'32"S, 43 degrees 37'35"W), State of Rio de Janeiro, Brazil, between January 2008 and March 2008. The male copulatory organ of L. tainhae n. sp. differs from the all known species of this genus in having the largest accessory piece, the length of accessory piece exceeding the length of the copulatory organ tube, and the distal tip of the lower lobe crossing the upper lobe. Ligophorus brasiliensis n. sp. and L. guanduensis n. sp. have a similar shape of the accessory piece, but in L. guanduensis n. sp. the lower lobe is larger than the upper lobe (as opposed to L. brasiliensis n. sp.), the ratio between length of upper lobe and the length of the proximal part of the accessory piece before the bifurcation is shorter and the distal tip of the lower lobe extends to the level of the upper lobe (in L. brasiliensis n. sp. the distal tip of lower lobe crossing the upper lobe). In L. lizae n. sp., the terminal bifurcations of the accessory piece are equal in length and unequal in the other 3 new species. Species of Ligophorus are recorded for the first time from Brazil.

The hypoosmotic swelling test in fresh rabbit spermatozoa.

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil.

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.

Evaluation of glucose as a cryoprotectant for boar semen.

Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111.0 mM Tris, 31.4 mM citric acid, 185.0 mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200 mM Tris; 70.8 mM citric acid, 55.5 mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200 mM Tris, 70.8 mM citric acid, 55.5 mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30 degrees C/minute to -196 degrees C. The straws were thawed at 60 degrees C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively

Assessment of sperm morphology in zebu bulls, under field conditions in the tropics.

Sperm morphology was studied in 302 extensively managed Zebu bulls (aged 1.5-9 years), classified as sound (n=166) or unsound (n=136) for breeding, under field conditions in the dry tropics of Costa Rica. Single semen samples were collected by electro-ejaculation and fixed in formol-saline solution immediately after collection. Sperm morphology was determined in the field on wet smears using a microscope equipped with phase-contrast optics, and further determined in the laboratory on air-dried smears stained with carbol-fuchsin. The frequencies of sperm abnormalities (such as abnormal acrosome, head, neck, mid-piece, tail, and presence of cytoplasmic droplets) were recorded as a percentage of the total number of counted spermatozoa (400 cells). Zebu bulls considered unsound for breeding showed a higher mean prevalence (p < 0.05) of knobbed acrosomes (4.0 versus 0.9%), head defects [specifically, nuclear invaginations and heads with abnormal shapes and sizes (27.6 versus 4.0%)], abnormal tails (11.2 versus 4.7%), and proximal droplets (8.4 versus 1.6%), compared with bulls considered sound for breeding. In these latter bulls, the abnormality most commonly seen was the presence of single bent tails with an entrapped cytoplasmic droplet (3.0 +/- 3.7%). Young Zebu bulls (i.e. bulls under 2 years of age) showed a higher percentage of missing acrosomes, and proximal cytoplasmic droplets, than older sires (12.1 versus 2.4%, and 23.9 versus 3.6%, respectively; p < 0.05), interpreted as an indication of low ejaculation frequency and sexual immaturity, respectively. Bulls with a long scrotum and soft testicular consistency (TC) at palpation showed higher percentages of abnormal sperm heads in the ejaculate than bulls with a normal scrotal length (SL) and a normal TC (32.7 versus 12.8% and 30.7 versus 10.3%, respectively; p < 0.05). In addition, Zebu bulls with a scrotal circumference (SC) < or = 30 cm showed a higher prevalence of proximal cytoplasmic droplets than bulls whose SC was > 30 cm (9.8 versus 2.6%, p < 0.05). A higher mean percentage of abnormally sized and shaped heads, especially undeveloped and narrow at the base, was more frequently found in stained smears than in unstained samples (26.0 versus 9.9%, p < 0.05), which clearly underlines the importance of using both stained and wet smears when assessing sperm head morphology. However, for a quick assessment of sperm morphology under field, tropical conditions, phase-contrast microscopy provides useful information for the spermiogramme evaluation.