Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

Histochemical study of the oviducal gland and analysis of the sperm storage tubules of Mustelus schmitti Springer, 1939 (Chondrichthyes, Triakidae)

Abstract: The paired oviducal glands of immature and mature females of Mustelus schmitti were examined macro and microscopically. Findings indicate that these glands possessed the same zonation as in most chondrichthyans from anterior to posterior: club, papillary, baffle and terminal zones. The whole gland is composed by simple tubular glands that connect with transverse grooves all along the organ. The club zone presents a typical indian club shape with a simple columnar and ciliated epithelium including secretory cells PAS (+) and AB (+). The papillary zone is characterized by lamella forming small and long cones in numbers of three. The epithelium of this zone contains ciliated cells with apical nuclei and secretory cells with basal nuclei that stain AB (+)The baffle zone consists of apically flattened lamellae alternating with spinnerets which are small projections disposed by both sides of the plateau. This whole structure is present in number of 8 or 9 units. A simple columnar ciliated epithelium covers the plateau and spinnerets and no AB or PAS staining is observed. The epithelium of the terminal zone is PAS (-) and AB (+), and elongated tubules, that run adjacent to the baffle zone are the site where groups of spermatozoa are clearly observed in the lumen. The epithelium of the sperm storage tubules do not stain with any of the dyes tested. Sperm was also observed in the baffle zone, presumably in its way to the fecundation in the oviduct because it displays no aggregation pattern and was between the folds of the epithelium. By scanning electron microscopy sperm was observed in the club and baffle zones in a gland which belonged to a pregnant female.

Proteolytic release and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control, NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

Novel genes and regulatory systems in epididymal differentiation and sperm maturation of the mouse.

Mammalian spermatozoa gain their fertilizing ability during maturation in the epididymis. Proteins and lipids secreted into the epididymal lumen remodel the sperm membrane, thereby providing the structure necessary for progressive motility and oocyte interaction. In the current study, genetically modified mouse models were utilized to determine the role of novel genes and regulatory systems in the postnatal development and function of the epididymis. Ablation of the mouse β-defensin, Defb41, altered the flagellar movements of sperm and reduced the ability of sperm to bind to the oocyte in vitro. The Defb41-deficient iCre knock-in mouse model was furthermore utilized to generate Dicer1 conditional knock-out (cKO) mice. DICER1 is required for production of mature microRNAs in the regulation of gene expression by RNA interference. Dicer1 cKO gave rise to dedifferentiation of the epididymal epithelium and an altered expression of genes involved in lipid synthesis. As a consequence, the cholesterol:polyunsaturated fatty acid ratio of the Dicer1 cKO sperm membrane was increased, which resulted in membrane instability and infertility. In conclusion, the results of the Defb41 study further support the important role of β-defensin family members in sperm maturation. The regulatory role of Dicer1 was also shown to be required for epididymal development. In addition, the study is the first to show a clear connection between lipid homeostasis in the epididymis and sperm membrane integrity. Taken together, the results give important new evidence on the regulatory system guiding epididymal development and function

Minor sperm abnormalities in young male post-pubertal patients with juvenile dermatomyositis

The objective of the present study was to identify sperm abnormalities in young male patients with juvenile dermatomyositis (JDM). In 2005, 18 male JDM patients, diagnosed according to the criteria of Bohan and Peter, were followed at the Pediatric Rheumatology Unit and Rheumatology Division, of our Institution. Of the 18 males, 11 were pre-pubertal and 7 were post-pubertal. Two of 7 post-pubertal JDM male patients were excluded: one for orchidopexy for cryptorchidism and the other for testicular ectopia in the left testis. The remaining 5 post-pubertal JDM patients were prospectively evaluated on the basis of two semen analyses, according to the World Health Organization (WHO), urologic evaluation, testicular Doppler ultrasound hormone profile. The data of the JDM patients were compared with those of 5 age-matched healthy controls. The median age 18, was similar in JDM patients and controls. All JDM patients had teratozoospermia (abnormal sperm morphology), as did 4 (80%) of the controls. One of JDM patients had previous oligoasthenoteratozoospermia treated with intravenous cyclophosphamide with normalization of the number and concentration of the sperm after 5 years. All sperm parameters (sperm concentration, total sperm count and total motile sperm count by WHO, and sperm morphology by Kruger strict criteria), testicular volumes by Prader orchidometer and ultrasound, and hormones were similar in JDM patients compared with controls. The frequency of anti-sperm antibodies was similar in both groups. All JDM patients had minor sperm abnormalities in the head, midpiece, and/or tail of spermatozoids. Serial semen analyses in larger study populations are necessary to identify the extent and duration of sperm abnormalities in male patients with idiopathic inflammatory myopathies.

Reproduction in the temperate crayfish, Orconectes rusticus: intermale aggression, copulatary behaviour, and sperm competition

The chelipeds of Orconectes rusticus are sexually dimorphic; males possessing the larger. Males use their chelae in intermale aggressive interactions, both to threaten, and assault opponents. In dyadic interactions males with larger chelae were dominant over otherwise physically similar opponents. A high frequency of attack behaviour, coupled with a low frequency of threats during these interactions indicates that actual physical contact is required for opponent assessment. Large clawed males oriented females into the copulatory position faster than small clawed males. Females more frequently escaped the precopulatory-grasp attempts of small clawed males. Additionally, male-female pairs that included a large clawed male remained in copula longer than pairs that included a small clawed male. Sperm of the second male to mate took precedence over the sperm of the primary male. Sperm precedence was incomplete; about 900/0 paternity accrued to the second male.

Sperm utilization patterns in Gryllus integer (orthoptera: gryllidae

Sperm competition is the competition for fertilizations between ejaculates, within a female, following multiple mating. There are four sperm utilization or precedence patterns: first male precedence, where the first male to mate fertilizes most of the eggs laid by a female; last male precedence, where the last male to mate fertilizes most of the eggs laid by a female; "all-or-none" pattern, where sperm from either male fertilizes all the eggs laid by a female but which male's sperm that is used is random; or sperm mixing, where sperm from each male is used equally in fertilizing eggs laid by a female. Intermediate utilization patterns are also possible. Sperm competition occurs in a wide variety of insect species as well as other animals. This study was undertaken to study sperm competition in the field cricket, Gryllus integer. Four experiments were conducted: a radiation and sterilization experiment, a diapause experiment, and 2 competition experiments. It was found that 7,000 rad of gamma radiation sterilized adult ~ integer males. There was no diapause in the laboratory in ~ integer eggs. In the first competition experiment, three groups of females were used: females mated with a normal male, then with a second normal male (NN group); females mated with a normal male, and then with a sterile male (NR group); and females mated with a sterile male, and then with a normal male (RN group). The results obtained from this experiment showed that the mean proportion of eggs hatched was significantly different between 3 groups of females, with the proportion hatched much greater in the NN group than in either the NR or RN groups. The pattern for the proportion of eggs hatched following a double mating most closely resembled a pattern expected if sperm mixing is occurring. Results obtained in the replicate competition experiment showed that the mean proportion of eggs hatched for the females in the NR group was significantly lower than the proportion hatched in the other two groups. This also supports a model of sperm mixing as a precedence pattern. Values calculated following Boorman and Parker (1976), for the proportion of eggs fertilized by the second male to mate following a double mating, were 0.57 in competition experiment 1 and 0.62 in the replicate. These values indicate that sperm mixing occurs in~ integer.

Intraovarian sperm storage in Helicolenus dactylopterus dactylopterus fertilitzation, crypt formation and maintenance of stored sperm

The females of the bluemouth rockfish, Helicolenus dactylopterus dactylopterus (DelaRoche, 1809), store sperm within their ovaries for periods of up to 10 months. Twenty six females with standard lengths between 152 and 257 mm and six males with standard lengths between 253 and 209 mm were caught storage crypts with stored spermatozoa and to describe their evolution over the year. After internal fertilization and once sperm reaches the ovary, a crypt forms probably by an epithelial inclusion at the base of the lamellae of one or several spermatozoa groups that are floating freely in the interlamellar space of the ovarian lumen. Stored spermatozoa have a large cytoplasm bag surrounding their heads. This bag could serve as a nutritive reservoir during the long storage period. Many desmosonal and tight junctions between the crypt cells ensure tha male sex cells are protected against the female immune system

Spermiogenesis particularities of a sperm storage species: Helicolenus dactylopterus (Teleostei: Scorpaenidae)

A study of the spermiogenesis and spermatozoa of Helicolenus dactylopterus was conducted. Females of this species have the capacity to store sperm within their ovaries, and male gametes have a considerable cytoplasmic mass surrounding their heads to survive the long period of intraovarian sperm storage. Our observations show that early spermatids are round-shaped cells and have a spherical nucleus with diffuse chromatin. The nuclear volume decreases as a result of progressive chromatin condensation during spermiogenesis, causing the nucleus to take on a U-shape. Flagellar insertion is not central to the nucleus but consistently occurs at an oblique angle towards one side of it. The flagellum is inserted into the nuclear fossa, without subsequent nuclear rotation. In mature spermatozoa, the flagellum is adjacent to the nucleus. A comparison of the spermatozoa in the testicular lobules and those in the intraovarian storage structures suggests that the increase in volume of the cytoplasmic mass may occur in the posterior region of the testis, in the testicular duct. Spermatozoa enter the ovary in groups that reach the ovarian lumen and are surrounded by the ovarian epithelium for storage in sperm storage crypts

A practical approach on boar sperm cryodamage. Morphofunctional and immunocytochemical study of cryopreserved boar sperm intended for use in artificial insemination

L'ús d'esperma criopreservada en la inseminació artificial (IA) d'espècies d'interès productiu permet un major control sanitari i la creació de bancs de germoplasma d'alt valor genètic, entre d'altres avantatges. En el mercat porcí la major part de les inseminacions són encara realitzades amb semen refrigerat degut a l'èxit de l'aplicació de diluents de llarga durada i també a causa de la sensibilitat de l'esperma porcina a la criopreservació. Malgrat que aquesta sensibilitat ve donada per característiques particulars de la fisiologia espermàtica en l'espècie, algunes ejaculacions mantenen els paràmetres de qualitat espermàtica després de la criopreservació (ejaculacions amb bona "congelabilitat", GFEs) enfront d'altres que no sobreviuen al procés (ejaculacions amb mala "congelabilitat", PFEs). El primer objectiu de l'estudi va ser comparar ambdós grups en termes de fertilitat in vivo. El segon objectiu va ser testar l'eficiència de la inseminació postcervical (post-CAI) amb l'esperma criopreservada. El tercer objectiu va ser buscar predictors de la congelabilitat de les ejaculacions, tant en les GFEs com en les PFEs i en tres passos del procés de criopreservació (a 17ºC, a 5ºC i a 240 min postdescongelació). Aquest objectiu es va dur a terme mitjançant l'avaluació de paràmetres convencionals de qualitat espermàtica i a través de l'estudi de la localització i la reactivitat sota el microscopi de tres proteïnes (GLUT3, HSP90AA1 i Cu/ZnSOD) relacionades amb la fisiologia espermàtica i amb possibles rols en la congelabilitat. El quart objectiu va ser quantificar l'expressió de les tres proteïnes per transferència western, tant en espermatozoides d'ejaculacions GFEs com en els d'ejaculacions PFEs i en els tres passos abans esmentats, per tal de determinar el seu potencial com a predictores de la congelabilitat. Pel primer i el segon objectiu, 86 truges van ser inseminades per post-CAI amb 26 ejaculacions de mascles Piétrain dividides en una porció refrigerada a 17ºC (tractament control) i una porció criopreservada, ambdues porcions classificades alhora com a GFEs o PFEs. Els resultats més rellevants van demostrar que les probabilitats d'embaràs eren dues vegades menors en inseminacions amb esperma criopreservada d'ejaculacions PFEs (P < 0.05) que en inseminacions amb esperma criopreservada d'ejaculacions GFEs, fet que indica que les ejaculacions amb percentatges elevats d'espermatozoides mòbils progressius i d'integritat de membrana (per sobre del 40% en les GFEs) són més favorables a provocar embarassos que no pas aquelles ejaculacions amb una pobra funció espermàtica in vitro (PFEs). Ni el nombre de truges que van donar a llum, ni la quantitat de garrins, ni el risc de reflux espermàtic van ser significativament diferents entre les inseminacions amb esperma criopreservada d'ejaculacions GFEs i les inseminacions control amb semen refrigerat, la qual cosa demostra la bona aplicabilitat de la inseminació post-CAI amb l'esperma criopreservada. Finalment, pel tercer i quart objectius van ser criopreservades 29 i 11 ejaculacions de mascles Piétrain, respectivament. Dos paràmetres cinètics espermàtics, la linealitat (LIN) i la rectitud (STR), van mostrar una hiperactivació de la mobilitat superior en les ejaculacions PFEs que en les GFEs després de 30 min a 5ºC durant la criopreservació. A més, la combinació d'ambdós paràmetres va donar una fiabilitat propera al 72% en la predicció de la congelabilitat de les ejaculacions porcines. Tot i que no va ser possible predir la congelabilitat mitjançant l'avaluació de les tres proteïnes al microscopi, els resultats de transferència western van revelar diferències en l'expressió de la HSP90AA1 en l'esperma a 17ºC, molt possiblement relacionades amb la millor supervivència a la criopreservació dels espermatozoides d'ejaculacions GFEs. Aquests resultats suggereixen que la promoció de la criopreservació d'esperma porcina per la seva aplicació en IA passa pel desenvolupament de tests per la predicció de la congelabilitat en semen refrigerat.

New insights into boar sperm function and survival from integrated field and laboratory studies

En aquesta tesi s'han dut a terme dos tipus d'estudis diferents. L'objectiu del primer era la preservació del semen de porcí a 15ºC i el del segon eren els co-cultius homòlegs de cèl·lules epitelials de l'oviducte i espermatozoides de porcí. Pel que fa al primer estudi, s'ha observat que l'addició de la prostaglandina F2α i àcid hialurònic a les dosis seminals no malmena la qualitat espermàtica i que la tolerància dels espermatozoides als canvis d'osmolalitat del medi es pot correlacionar proves de fertilitat i prolificitat.. Respecte el segon, s'ha determinat que les cèl·lules oviductals afecten els paràmetres espermàtics i que la presència d'espermatozoides sobreexpressa els gens que codifiquen per les proteïnes de xoc tèrmic. Així, se suggereix que aquestes proteïnes tenen algun paper en els processos reproductius que tenen lloc a l'oviducte, malgrat que s'hagi observat, mitjançant la tècnica de la interferència de l'RNA, que la HSP90AA1 no està implicada en el perllongament de la viabilitat espermàtica.

Sperm competition and the evolution of testes size in terrestrial mammalian carnivores

Summary Understanding the factors influencing variation in the degree of sperm competition is a key question underlying the mechanisms driving sexual conflict. Previous behavioural and comparative studies have indicated that carnivores appear to have evolved under sperm competition but an analysis of the predictors of the level of sperm competition is missing. In this study, we use phylogenetic comparative methods to investigate life-history parameters predicted to affect the degree of sperm competition in terrestrial carnivores using variation in relative testes size (RTS, after controlling for body size allometry) as a measure of the level of sperm competition. Due to a paucity of consistent data across taxa, we used three measures of RTS: testes mass (n = 40 species), testes and epididymes mass combined (n = 38), and testes volume (n = 48). We also created a derived data set (n = 79) with testes mass estimated from regression analyses on the other measures of testes size. Carnivores with shorter mating seasons had relatively larger testes, consistent with the hypothesis that sperm competition is greater when the degree of female oestrous synchrony is high. This relationship was stronger in spontaneous versus induced ovulators, suggesting higher sperm competition levels in spontaneous ovulators. This is the first comparative study to show this within mammalian taxa. Neither social mating system nor reproductive lifespan were significantly associated with variation in RTS and hence are poor predictors of sperm competition levels. None of the above relationships were found to be significant for the testes and epididymes mass combined data set, but our understanding of the role of the epididymis in sperm competition is too limited to draw any conclusions. Finally, we consistently found a significant phylogenetic signal in all analyses, indicating that phylogeny has played a significant role in the evolution of carnivore testes size and, therefore, in shaping levels of sperm competition. Our results shed new light into the factors affecting levels of sperm competition in terrestrial carnivores by showing that the degree of oestrous synchrony and ovulation type interact to predict variation in RTS.