Postspawning Destruction of Male Gametes in Polycyclic and Monocyclic Bony Fishes: Pointhead Flounder, <Emphasis Type="Italic">Cleisthenes herzensteini</Emphasis> Schmidt (Pleuronectidae) and Chum Salmon, <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> Walbaum (Salmonidae)

The process of the destruction of nonreleased spermatozoa was studied using TEM in the postspawning gonads of the chum salmon Oncorhynchus keta and the pointhead flounder Cleisthenes herzensteini, as representatives of monocyclic and polycyclic fishes respectively. It was shown that in both species residual sperm cells are destroyed through fragmentation of chromatin and the entire cell. It is supposed that the process of destruction in this case is connected with autolytic disintegration known in some multicellular animals, but never described for Teleostei.Original Russian Text Copyright © 2005 by Biologiya Morya, Neznanova, Ivankov, Reunov.     

Structures related to the germ plasm in mouse

This report presents data from ultrastructural and morphometric studies on the germinal-body-like structures, nuage, nuage-mitochondrial clusters and chromatoid bodies in 4.5-day embryo cells and spermatogenic cells of the laboratory mouse Mus musculus. In the 4.5-day embryo cells the germinal-body-like structures that, according to previous data, arise by condensation of mitochondria in Graafian oocytes, were found not to undergo any ultrastructural alterations. In spermatogonia the germinal-body-like structures presumably were transformed into nuage that functioned as 'intermitochondrial cement' binding the mitochondrial clusters. In primary spermatocytes mitochondria aggregated by nuage were found with large vacuoles containing membraneous conglomerates that were obviously excreted by organelles into the cytoplasm. The chromatoid bodies that arose in spermatocytes and finally disintegrated in the posterior part of late spermatids seemed not to be implicated in the pathway of the germinal-body-like structure. The dispersion of chromatoid bodies was noted to be accompanied by excretion of membraneous conglomerates by late spermatid mitochondria. The spermatozoa were not found to contain either the germinal-body-like structures or any other germ-plasm-related structures.     

The ultrastructure of the peculiar synspermia of some Dysderidae (Araneae, Arachnida)

The present study reports on the ultrastructure features of spermatozoa and spermatogenesis of several species of Dysderidae (Dysdera crocata, Dysdera erythrina, Dysdera ninnii, Harpactea arguta, Harpactea piligera, Dasumia taeniifera). Dysderid spiders are known to possess a peculiar sperm transfer form known as synspermia, characterized by fused spermatozoa surrounded by a secreted sheath. Until now the exact mode of formation of the synspermia is unknown. The present study demonstrates that the spermatids are connected via narrow cell bridges during the entire spermiogenesis as is usual, although in Dysderidae they do not separate at end of the spermiogenesis. Instead, they fuse completely within the testes shortly after the spermatid has coiled to get a spherical shape. The number of fusing sperm cells is different in the different observed species. The species of the genus Harpactea thus have synspermia consisting of two fused spermatozoa; whereas in the species of the genus Dysdera four sperm cells are fused and in D. taeniifera at least three spermatozoa are fused. In contrast with other known families with this peculiar form transfer of sperm, the synspermia in Dysderidae are mainly characterized by a conspicuous vesicular area which extends through the entire synspermium surrounding the cell organelles. Thus, all main cell components (e.g., nucleus, acrosomal vacuole, and axoneme) are covered by the vesicular membrane. The vesicular area seems to be functional and probably it is important during sperm activation in female genital system. Simultaneously to the extension of the vesicular area, the synspermium accumulates large amounts of glycogen. The glycogen is mainly located around the centriolar adjunct and along the axoneme accompanying the postcentriolar elongation of the nucleus. A further peculiar feature is the extremely elongated acrosomal vacuole, which seems to be synapomorphic trait for sperm cells of dysderids. Interestingly, spermatogenesis, including the fusion, exclusively occurs within the testes (in contrast to the formation of coenospermia). In the vas deferens only synspermia were found. The secreted sheath surrounding the spermatozoa is finally synthesized in the parts of the vasa deferentia, which are close to the genital opening where numerous vacuoles and microvilli are seen in the epithelial cells.     

Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.     

On the morphology of spermatozoa of tuco-tucos, Ctenomys (Rodentia: Ctenomyidae): New data and its implications for the evolution of the genus

Sperm morphology was studied in 10 species of the caviomorph rodent Ctenomys. Ctenomys argentinus, C. conoueri, C. dorbigny and C.perrensis had symmetric spermatozoa with paddle-like heads. Ctenomys australis, C. mendocinus, C. porteousi, C. rionegrensis and Ctenomys sp., on the other hand, had spermatozoa with paddle-like heads but with the tail inserted at one side of the central axis and a nuclear caudal extension originating from the base of the head at the opposite side of the insertion of the tail and running parallel to the flagellum; these spermatozoa are referred to as simple-asymmetric. In C. yolandae, a complex-asymmetric morphological type not previously described for the genus was found. This type is characterized by the presence of two nuclear caudal extensions. Symmetric spermatozoa (total length = 52 pm) were shorter than asymmetric (both simple and complex) ones (total length = 87 pm). In spite of these differences, the relative size of heads, midpieces and tails were maintained in the three groups, representing 12%, I I YO and 88% of the average total length, respectively. Within each group of species bearing the same sperm type, a low interspecific variability both in morphological patterns and dimensions of sperm cells was observed. This low interspecific variability associated with the north to south geographical distribution of species having, respectively, symmetric and asymmetric spermatozoa, suggests that these characters appeared at an early stage in evolution of the group, and probably played an important role in the first steps of speciation by promoting reproductive isolation.     

Ultrastructure of the male genital tract,spermatogenesis and spermatozoa of hattena cometis domrow (Acari: Gamasida: Ameroseiidae)

The ameroseiid mite Hattena cometis has a male genital system that consists of an unpaired, u‐shaped testis and paired deferent ducts leading into an unpaired accessory genital gland and ejaculatory duct. The genital opening is located anteriorly immediately in front of the sternal shield. Spermatogenesis is simple, probably due to the haploid nature of the male. Eight stages of spermatogenesis could be roughly distinguished. Mature spermatozoa as found in the deferent duct lumen are peculiar in having a bisected nucleus and numerous peripheral flat chambers, which were formed from indentations of the plasmalemma. In inseminated females, spermatozoa were observed in the syncytial tissue of the sperm access system and in the somatic cells of the ovary. These spermatozoa have achieved a new structure, i.e., an electron‐dense plate dividing the cell into two unequal halves. The dense plate has an intricate substructure. Its function is unknown. These sperm cells are considered to represent capacitated spermatozoa. The peripheral chambers are reduced in number inside the female. Similar sperm cells, containing a dense plate, were seen in vacuoles within the epithelium of the deferent duct of one male. These cells are evidently under destruction, but before being completely dissolved had undergone a development leading beyond that of the mature sperm cells found in the deferent duct. Apparently, entering the cell of the deferent duct epithelium or the syncytium tissue triggers the production of the dense plate (or the capacitation process). Our observations are compared with results obtained from other anactinotrichid Acari, mainly Gamasida, and confirm and complete the interpretation of the correlated evolution of components of gamasid reproductive systems. J. Morphol. 274:1010–1025, 2013. © 2013 Wiley Periodicals, Inc.     

IN VITRO FERTILIZATION OF IN VITRO MATURED MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) FOLLICULAR OOCYTES

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales ( Balaenoptera acutorostrata ) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro-nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at - 80°C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher ( P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration ( P < 0.05) and pronuclear formation ( P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.     

Localization of a single sperm membrane autoantigen (RSA-1) on spermatogenic cells and spermatozoa

The rabbit sperm membrane autoantigen RSA-1 is a sialoglycoprotein of 13,000 daltons which first appears on the surface of pachytene spermatocytes. Using specific antiserum to RSA-1 the antigen has been localized by immunofluorescence and immunoperoxidase staining. On testicular cells labeled at 37°C, RSA-1 is seen in patches on the surfaces of pachytene spermatocytes, round spermatids, and over the acrosomal area of later spermatids and spermatozoa. Over the postacrosomal and middle-piece regions of late spermatids and spermatozoa the labeling appears uniform. The uniformity can be seen to stop abruptly at the equatorial segment-postacrosomal border. Labeling cells after fixation gives a uniform distribution of label over the surface where patches were seen at 37°C. The polypeptides recognized by the antiserum used for labeling were identified by immunoadsorbent chromatography and subsequent SDS-PAGE. In testicular cells anti-RSA-1 recognizes the 13,000-dalton form and another component which migrates with the dye front. In ejaculated spermatozoa anti-RSA-1 recognizes a distinct ejaculate complex of higher-molecular-weight proteins containing an 84,000-dalton major band and five minor components.     

Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay

Defective mammalian spermatozoa become ubiquitinated during epididymal passage, a mechanism that may mark the abnormal spermatozoa for proteolytic destruction (Sutovsky et al., 2001a: J Cell Sci 114:1665-1675). It is not known how such spermatozoa are recognized by the epididymal ubiquitination pathway and whether there is a selection against certain types of sperm defects. We examined the relationship between sperm ubiqutination, lifelong sperm morphology and sperm DNA defects using a single chanel, ubiquitin-activated flow cytometric assay, and a dual, ubiquitin-TUNEL assay. Semen samples from nine service sires of good-to-average fertility were screened. A positive correlation was found between sperm ubiquitination and the average frequency of morphological semen abnormalities from field evaluations performed throughout the reproductive life of individual sires. Sample correlation coefficients were r=0.65 for primary (head and tail) and r=0.60 for total semen abnormalities in the single channel assay. In a dual assay, we found a high, positive correlation (r=0.93) between the ubiquitin-positive sperm and the TUNEL positive sperm. Substantial correlations (r=0.47-0.64) were observed when the measurements from these two respective assays were compared for individual sires. While anti-ubiquitin antibodies recognized most of the TUNEL-positive sperm cells, the TUNEL-positive spermatozoa represented only a subset (approximately 20-40%) of all ubiquitin-positive cells. It appears that the ubiquitin-dependent sperm quality control, residing in the epididymal epithelium, has the ability to detect spermatozoa with apoptotic or necrotic DNA, while spermatozoa with defects other than DNA fragmentation are also recognized and ubiquitinated.     

Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.     

On the ecology and distribution of Pseudoterranova decipiens C (Nematoda: Anisakidae) in an intermediate host, Hippoglossoides platessoides, in northern Norwegian waters.

The distribution of Pseudoterranova decipiens C helps demonstrate the ecological basis of this genetically defined sibling species. In northern Norwegian waters the major fish intermediate host is Hippoglossoides platessoides. Overall prevalence, mean intensity and intensity range in H. platessoides were 15%, 16.5 and 1-165, respectively. Outside the range of its only known definitive host, the seal Erignathus barbatus, the parasite was not found in the same intermediate host.     

Effects of X-irradiation on the kinetics of abnormal sperm production and sperm loss in the mouse

Exposure of male mice to 6 Gy of X-rays resulted in a very rapid and extensive sloughing of the germinal epithelium as shown by the accumulation of non-sperm cells within the lumen of the epididymis. These cells were identified as stage 1 and 2 round spermatids. After accumulating in the caput, they progressed through the epididymis over the weeks of sampling and, by Week 9 after irradiation, they had completely disappeared from the organ. It is suggested that the precocious loss of round spermatids is responsible for the induction of oligospermy within the testis and the caput epididymidis. Similar sperm losses from the cauda epididymidis were not observed. Radiation also enhanced the frequency of misshapen spermatozoa normally found in this strain. From kinetic considerations, it is suggested that the generation of abnormal spermatozoa may be biphasic with an early component comprising maturing spermatids and a late contingent composed of affected spermatocytes. Return to the pre-irradiation level of abnormal frequency was not observed within the time frame of this study (10 weeks), perhaps indicating residual damage. The synchrony that existed among the various organs in terms of both sperm loss and the generation of abnormal spermatozoa may be the result of a rapid dispersion of gametes from the testis and not due to local responses as would be expected if sperm flow were affected by the irradiation. The distribution of abnormal sperm types was different in the testis from that in the epididymis, presumably because of a testicular spermatophagic mechanism specific for the removal of certain deformities. It is concluded that the kinetics of spermatogenesis, of spermiogenesis, and of sperm transport in the mouse is not affected by exposure to 6 Gy of X-rays.     

Electron microscopy of the skin of the teleost,Hippoglossoides elassodon

Summary The normal skin of the pleuronectid fish, Hippoglossoides elassodon, is described by light and electron microscopy. The epidermis consists of 5 to 9 layers of cells, the majority of which are squamous cells and the minority mucous cells. The squamous cells are characterized by numerous desmosomes and associated cytoplasmic filaments. The mucous cells accumulate mucous droplets in vacuoles of Golgi origin and are observed apparently in the process of releasing their content at the free surface. The dermis consists of alternating lamellae composed of typical collagen fibers. Pigment cells are of three types: melanophores, iridophores (guanophores), and lipophores.This work was supported by Public Health Service Research Grant CA-08158 from the National Cancer Institute.     

Respiration of bull spermatozoa in presence of exogenous glutathione (GSH)

In the investigations of the rate of oxygen uptake by sperm cells in frozen bull semen it was found that exogenous glutathione (GSH) in a 5 mM concentration failed to stimulate mitochondrial oxidative processes in the sperm cells. At the same time it was observed that the respiration of sperm cells changed in relation to the season of the year when ejaculates had been collected and preserved. The highest values were noted in winter and the lowest ones in summer. Moreover, in the observations on the motility of spermatozoa a difference was observed in the reaction of sperm cells to exogenous GSH, and this difference depended on the season. A significant (p less than or equal to 0.05) rise was disclosed in the number of sperm cells with maintained advancing motility in the presence of GSH in the medium as compared to controls without GSH, but this rise was found only in semen obtained and preserved in winter.     

Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes

Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo-micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (gamma-tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P = 0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected.     

Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? cytochemical studies

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Bovine oviductal fluid (bOF) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in vitro

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO₂. Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P < 0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P > 0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P > 0.05). Sperm PM integrity was not affected (P > 0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18–24 h) incubation periods without affecting sperm viability.