Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cell monolayers

Capacitation of fresh bovine spermatozoa on bovine epithelial oviduct cells was assessed1) by the ability of spermatozoa to fertilize bovine oocytes in vitro and2) by exposure to lysophosphatidylcholine (LC) to induce acrosome reaction in the capacitated spermatozoa. When spermatozoa were incubated on bovine epithelial oviduct cells in B2 medium supplemented with 10% estrous cow serum (ECS) and then exposed to 100 mug/ml LC for 15 minutes, the percentage of acrosome reaction induced increased in a time-dependent course, reaching a plateau after 6 hours. Inversely, when spermatozoa were incubated in B2 + 10% ECS alone, the percentage of acrosome reaction induced by LC didn't fluctuate. The in vitro fertilization rate obtained after incubation of spermatozoa during 6 hours on bovine epithelial oviduct cells in B2 + 10% ECS medium was on average 75% for both the preovulated and ovulated oocytes. The developmental stages observed 18 hours after male and female gamete co-culture were similar to those obtained after in vivo fertilization. This study suggests that incubation of fresh bovine spermatozoa on bovine oviduct epithelial cell monolayers during 6 hours is an efficient method, and one that is close to in vivo capacitation.     

Effects of bovine oviductal proteins on bull spermatozoal function

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.     

Heparin and Ca2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers

The success of assisted reproductive techniques, such as IVF, could be enhanced by being able to select the most competent spermatozoa in a sample. Attachment and subsequent release of spermatozoa from oviductal epithelial cells (OEC) could provide populations of functionally superior spermatozoa for use in these protocols. The objective of the present study was to investigate the ability of heparin and Ca2+-free medium to induce spermatozoa release from bovine OEC. Epithelial cells were grown to confluence in 24-well plates and pooled frozen bull semen was added to a final concentration of 1 x 10(6) spermatozoa/well. Spermatozoa were allowed to bind to OEC for 2 h. Medium with unbound spermatozoa was removed and replaced by Sperm-TALP, only (control), with heparin (5, 10, or 15 IU/mL), or Ca2+-free with 2 mM EGTA. Treatments were left on sperm-OEC co-cultures for 0.5, 1, 2, 3, or 5 h. At each time, the media were recovered and spermatozoa from each treatment were counted and evaluated for acrosome integrity and motility. The total number of spermatozoa attached to OEC after 2 h of co-culture was considered 100%. Spermatozoa release is expressed as percentage of the total number of sperm cells bound to OEC after 2 h of co-culture. Data were analyzed by ANOVA and results are expressed as mean +/- SEM from three independent replicates. Beginning at 0.5 h, more sperm cells (P < 0.05) were released from OEC in the heparin groups (10 and 15 IU/mL, 77.3 +/- 6.2% and 84.0 +/- 6.2%, respectively) as compared to the control (46.4 +/- 6.2%). The Ca2+-free medium also induced spermatozoa release when compared with the control, but the effect was not significant until 3 h (38.2 +/- 1.9% vs 59.5 +/- 6.9%; P < 0.05). The percentage of acrosome reacted spermatozoa was not affected by heparin treatment. Heparin at 10 IU/mL increased (P < 0.05) the percentage of motile spermatozoa, whereas Ca2+-free medium caused the opposite effect at 0.5 h after addition of treatments. We conclude that both heparin and Ca2+-free medium are able to promote spermatozoa displacement from OEC attachment. Based on motility and acrosome status data, we predict that released sperm cells may be used for IVF and other assisted reproductive techniques.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

Behavior of bull spermatozoa in bovine uterine tube epithelial cell co-culture: an in vitro model for studying the cell interactions of reproduction

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.     

A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization

A non-activating semen diluent does not cause motility or acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of honey bee spermatozoa stored in ambient conditions were assessed 60 days pre-cryopreservation and 24 h post-cryopreservation. Seven variations of a Tris-based non-activating diluents (FEM1 – FEM7) were compared to samples treated with conventional activating diluent and untreated semen. Semen viability (membrane integrity) was assessed after short- and long-term storage at 14.0 ± 0.2 °C. The non-activating medium FEM7 contained more viable spermatozoa than the activating medium, 24 h after cryopreservation (67.6 ± 10.9% and ~4%, respectively). After 60 days, 22.0 ± 7.8% of spermatozoa was viable in non-activating medium versus 0.0 and 60.8 ± 12.3%, in conventional media and untreated controls, respectively. Hence FEM7 was used to cryopreserve bee semen and subsequently inseminate honey bee queens. The quality of brood produced by the queens was assessed 30–60 days after insemination. The percentage of worker-bee offspring (produced from successfully fertilized eggs) was ~75% for both the non-activating medium and the conventional extender medium. Our results indicate that a non-activating medium possesses significant advantage over the conventional activating medium if the semen requires storage after treatments such as cryopreservation. The percentage of female offspring (from fertilized eggs) produced by queens inseminated with semen diluted in either the activating or non-activating medium did not differ from one another.     

Prostaglandin E2, prostaglandin F2 alpha and endothelin-1 production by cow oviductal epithelial cell monolayers: effect of progesterone, estradiol 17 beta, oxytocin and luteinizing hormone

The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4] and estradiol 17 beta [E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10(4)/mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4; 1 microgram/mL), low P4 (L-P4; 10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL), OT (10(-9) M) ET-1 (10(-9) M), PGE2 (10(-8) M) PGF2 alpha (10(-9) M) or their combination (H-P4 + E2, L-P4 + E2, LH + E2, ET-1 + E2, L-P4 + E2 + LH and H-P4 + E2 + LH). The production of both PG and ET-1 was increased by E2 + low P4 and LH + E2 + low P4 (P < 0.05), while LH + E2 enhanced the production of PGF2 alpha and ET-1 (P < 0.05). Moreover, E2 + ET-1 stimulated PG production (P < 0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2 alpha and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids.     

Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa

Our laboratory has recently shown that in vitro-cultured oviductal cells secrete sperm motility maintaining factor(s). Since the binding of oviductal proteins to spermatozoa (SPZ) has been demonstrated in many species, the motility factor was postulated to bind the membranes of SPZ. Therefore, the current study was performed to evaluate which proteins from in vivo oviductal secretions bind to sperm membranes, to characterize binding conditions, and to evaluate the effect of this binding on sperm survival. Bovine oviducts were dissected, and oviductal cells and fluid were collected by pressing the oviductal tube with a glass slide. This mixture was incubated in Tris-EDTA buffer at 37°C for 30 min, and the cells were washed twice by centrifugation. The supernatant containing oviductal fluid proteins (OFP) was reserved, filtered, frozen (for later motility tests), or lyophilized and labeled with ¹²⁵|. Frozen-thawed SPZ were incubated either immediately, following capacitation, ionophore-induced acrosome reaction, death by heating, or flagellar removal with labeled OFP for 30 min. The resulting pellet after three washes was dissolved in SDS and submitted to 10% SDS-PAGE. An autoradiogram showed that 72, 66, 39, 38, and 36 kDa proteins bind strongly to the five types of SPZ used, and that this binding is very specific, since unlabeled OFP inhibited binding while serum proteins did not. Furthermore, for 39, 38, and 36 kDa proteins, the presence of calcium in the incubation medium was essential for dose-dependent binding, whereas magnesium was not. Preincubation of SPZ for 30 min at 37°C with oviductal fluid, followed by one wash and 6 hr of incubation in control media, showed that the percentage of motile SPZ is significantly higher (52 ± 6%) compared with SPZ not preincubated with oviductal fluid (24 ± 6%: P < 0.01). In summary, a limited number of proteins from oviductal secretions bind to the surface of bovine SPZ only in the presence of calcium, and this binding appears to be important for subsequent sperm viability. © 1996 Wiley-Liss, Inc.     

Effect of ovarian steroids and oxytocin on the production of prostaglandin E2, prostaglandin F2alpha and endothelin-1 from cow oviductal epithelial cell monolayers in vitro.

Cyclic physio-anatomical variation in the oviducts is mediated by the local countercurrent transfer of ovarian products. Thus, in this study cow oviductal epithelial cells (COEC) culture were utilized to investigate the effects of ovarian products such as progesterone (P4), estradiol 17beta (E2) and oxytocin (OT) on local oviductal prostaglandin E2 (PGE2), F2alpha (PGF2alpha) and endothelin-1 (ET-1) production. COEC were collected from non-pregnant Holstein cows (n = 8) during the follicular phase and cultured in M199 under standard culture conditions until monolayer formation. Cells in first passage were incubated for 24 or 48 h with P4 (500 ng/ml), E2 (1 ng/ml), OT (10(-9) M) or combination of E2 + P4. Administration of E2 significantly increased the production of PGE2, PGF2alpha and ET-1. However, simultaneous administration of P4 blocked the effect of E2. OT did not show any effect on oviductal productions of either PGs or ET-1. The results of this study show that E2 stimulates PG and ET-1 production by COEC in vitro. Thus, it can be suggested that locally transferred E2 from the ovarian follicles may be important for oviductal contraction and gamete/zygote transport during the peri-ovulatory period.     

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16°?C for 24?h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240?×?10⁶ spermatozoa/mL using animal protein–free commercial BIOXcell^® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell^® extender supplemented with buserelin acetate and stored 0, 8 and 24?h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell^® extender did positively affect the total motility (was higher in the observed samples with the addition of 2?µg/mL and 4?µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p?<?0.05) over the control group at the 0?h and 8?h of incubation following the supplementation of 2, 4 and 8?μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p?<?0.05) in 2?µg/mL and 4?µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4?µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4?µg/mL to the extender to improve the quality of bovine semen.     

Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.     

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.     

Effects of exposing bovine sperm to bovine serum albumin,or freeze-thawing,on sperm-bound amidase activity

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P < .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5°C or layered onto a solution of 6% BSA in extender at 37°C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5°C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P < .01). However, the amount of exposed sperm-bound amidase also was greater (P < .05) this was a deleterious change. Immediately after thawing, more (P < .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37°C there was no difference. After freezing and thawing of sperm, amidase activity was higher (P < .05) for the isolated sperm than for nonisolated cells. Thus, isolation of sperm using a 6% BSA gradient increased the proportion of progressively motile sperm, but decreased the percentage of sperm with an intact acrosome, based on measurements of amidase activity.     

Effects of benzyl alcohol on the bovine oestrous cycle and subsequent fertility

A uterine infusion varying in volume from 5 to 40 ml and containing from 0.5 to 2.0 ml of benzyl alcohol shortened the length of the oestrous cycle in cows treated in the early luteal phase and prolonged the oestrous cycle in some cows in the late luteal phase. Results with 399 infusions produced a concentration of cows in oestrus from 8 to 13 days after treatment. The conception rate for 198 inseminations at the first post-treatment oestrus was 59.6% compared to 59.2% obtained in 471 untreated cows.     

Effects of 4-n-nonylphenol and 17beta-oestradiol on early development of the barnacle Elminius modestus

Pollutants that are present in the aquatic environment and cause abnormal endocrine function in wildlife populations have been termed endocrine disrupting chemicals (EDCs). The impacts of these chemicals on the reproduction and development of vertebrates has been shown to be significant in both field studies and laboratory experiments. Over the past decade the number of investigations into the impacts of EDCs that affect reproductive and sexual characteristics (reproductive EDCs) has increased and evidence of their potency is evident in numerous wildlife species and through data from in vitro tests. However, little information is available on whether chemicals which act as EDCs in vertebrate species affect aquatic invertebrates. The case of imposex in archeogastropods following exposure to tributyltin (TBT) is a notable exception. Moreover, a number of studies have shown that development, fecundity and reproductive output of some aquatic invertebrates are affected significantly by exposure to pollutants. In order to determine whether external signs of exposure to vertebrate EDCs can be observed and monitored in invertebrate species, we exposed larvae of the barnacle Elminius modestus to environmentally realistic concentrations of the xeno-oestrogen, 4-n-nonylphenol (NP), and the natural oestrogen, 17beta-oestradiol (E(2)). Early life stages (nauplii and cyprids) were also exposed in the laboratory to determine whether there were effects on the timing of larval development and settlement. Ovary development and size of juveniles was measured following chronic exposure. Exposure to NP in the concentration range 0.01-10 μg l(-1) resulted in disruption of the timing of larval development. Similar results were obtained with E(2). Pulse exposures showed that the timing of exposure is critical and exposures for a period of 12 months caused long-term effects. A linear, concentration-dependent response was not evident.     

Effects of valerate on intestinal barrier function in cultured Caco-2 epithelial cell monolayers

Molecular Biology Reports - Short-chain fatty acids (SCFAs) are a group of microbial metabolites of undigested dietary fiber, protein and unabsorbed amino acids in the colon, well-known for their...     

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC)

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array? on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.     

Frozen-thawed bovine spermatozoa diluted by slow or rapid dilution method: measurements on occurrence of osmotic shock and sperm viability

This study was undertaken to investigate the occurrence of osmotic shock, sperm viability and membrane functional status of frozen-thawed bovine spermatozoa during a short-term incubation period (2 h) in vitro after dilution by 2 methods. Frozen semen from 10 bulls (0.5-ml plastic straws, 7% glycerol) was thawed and diluted by slow or rapid dilution method with Ham's F-10 medium containing 0 or 7% glycerol and assessed for sperm motion parameters, percentage of spermatozoa with coiled tails and reactivity to the hypoosmotic swelling (HOS; percentage of spermatozoa swelling) test at 60 min intervals during a 2 h incubation period (37 degrees C). Post-thaw sperm viability, as reflected by percentage and grade of motility (0 to 4) did not differ between the 2 dilution methods (P > 0.05) at the beginning of incubation (Time 0). However, differences were apparent (P < 0.05) as the incubation time increased. Slow dilution with medium containing 0% glycerol caused less increase (P < 0.05) in percentage of spermatozoa with coiled tails; Moreover, these spermatozoa showed greater reactivity to the HOS test. When contrasting slow vs rapid dilution methods, the occurrence of osmotic shock was less frequent, and response to the HOS test was greater for spermatozoa diluted slowly, regardless of the glycerol content of the incubation medium. Rapid deglycerolization of frozen-thawed bovine spermatozoa in a single step, induces damage which is not detected on the basis of spennatozoal motility but is clearly evident after several hours of incubation by using the HOS test to detect damage.     

Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP + BOEC; 3) in TALP + PHE; or 4) in TALP + BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.