Effect of homologous and heterologous seminal plasma on the fertilizing ability of ejaculated bull spermatozoa assessed by penetration of zona-free bovine oocytes.

The ability of seminal plasma to influence the fertility of ejaculated bull spermatozoa was examined using a sperm penetration assay for zona-free bovine oocytes. Washed, ejaculated spermatozoa from bulls of below (low) or above average (high) fertility were mixed with seminal plasma from the same bull, or with seminal plasma from a bull of contrasting fertility. Treated spermatozoa were stained with different fluorochromes and competed to penetrate oocytes after heterospermic insemination in vitro. Washed spermatozoa exposed to seminal plasma from bulls of high fertility penetrated more oocytes than those spermatozoa mixed with seminal plasma from bulls of low fertility (P < 0.01). Mixing low fertility spermatozoa with high fertility seminal plasma generally improved penetrating ability compared with low fertility spermatozoa mixed with low fertility seminal plasma (P = 0.05). Washed spermatozoa from a bull of low fertility mixed with his own seminal plasma had greater ability to penetrate oocytes than did washed spermatozoa from a bull of high fertility mixed with seminal plasma from a bull of low fertility (P < 0.01). The bias associated with using low fertility seminal plasma from the bull providing the spermatozoa was removed by repeating this experiment using pooled seminal plasma from different subfertile bulls. After combination with pooled seminal plasma from bulls of low fertility spermatozoa from bulls of high or low fertility penetrated oocytes in a similar way, but high fertility spermatozoa had a slightly higher penetration rate (P = 0.12). In conclusion, the penetration of zona-free oocytes by ejaculated spermatozoa from bulls of low fertility was marginally improved by seminal plasma from bulls of high fertility, but penetration by high fertility spermaotoza was decreased by exposure to low fertility seminal plasma. Seminal plasma from bulls of low fertility similarly affected the penetrating ability of high and low fertility spermatozoa if the seminal plasma used was foreign to the spermatozoa being tested.

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes.

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.

Effect of accessory sex gland fluid from bulls of differing fertilities on the ability of cauda epididymal sperm to penetrate zona-free bovine oocytes.

The ability of accessory sex gland fluid to affect the fertility of cauda epididymal sperm was evaluated for 10 bulls that ranged in fertility from 6.2% below to 6.0% above the average fertility of bulls at artificial breeding cooperatives. Cauda epididymal sperm collected from indwelling vasa deferentia catheters and cauda epididymal sperm exposed to accessory sex gland fluid from the same bull were compared on the basis of their rates of in vitro penetration of zona-free oocytes after heterospermic insemination. Incubation of cauda epididymal sperm with accessory sex gland fluid significantly enhanced the ability to penetrate oocytes, and bull fertility affected the magnitude of this improvement. For bulls of average and higher fertility, the positive influence of accessory sex gland fluid on penetrating ability of sperm was highly significant (p < 0.0001). Accessory sex gland fluid from bulls of below-average fertility also improved the penetrating ability of cauda epididymal sperm, although not significantly (p = 0.07). Heterospermic competitions compared the penetrating ability of cauda epididymal sperm exposed to homologous accessory sex gland fluid with a portion of the same sperm population incubated in heterologous accessory sex gland fluid from a bull of contrasting fertility. In experiments involving sperm from 12 different bulls, paired in 42 fertile/subfertile combinations, samples of cauda epididymal sperm mixed with accessory sex gland fluid from the higher-fertility bulls had greater oocyte-penetrating ability than when aliquots of that sample were mixed with accessory gland fluid from lower-fertility bulls (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol, phospholipid and phospholipase activity of ampullary and isthmic fluid from the bovine oviduct.

Cholesterol and phospholipid concentrations and phospholipase activity were measured in fluid from cannulae collected from the bovine oviductal isthmus and ampulla at different stages of the oestrous cycle. The cholesterol concentration and cholesterol normalized by protein were significantly (P = 0.03) greater in isthmic oviductal fluid (224.3 +/- 42.7 micrograms ml-1 over all stages) than in ampullary oviductal fluid (164.5 +/- 11.3 micrograms ml-1), and maximal concentrations (284.5 +/- 25.5 micrograms ml-1) were found during the luteal stage (serum progesterone concentration > or = 1.5 ng ml-1). The concentrations of the phospholipids sphingomyelin and lysophosphatidylcholine increased at different stages of the cycle and in different regions. In the ampulla, the concentration of sphingomyelin was significantly (P < 0.05) greater in oviductal fluid collected during the luteal phase (12.1 +/- 2.7% of total phospholipids) than in fluid collected near oestrus and ovulation (7.5 +/- 1.5% and 6.9 +/- 1%, respectively). The concentration of lysophosphatidylcholine was greater (P < 0.01) in ampullary (19.2 +/- 1.6% of total phospholipids) than in isthmic oviductal fluid (9.9 +/- 1.1%) collected near ovulation. The ratio of cholesterol to total phospholipid was highest in oviductal fluid collected from the isthmus during all stages (2.3 micrograms ml-1:% total phospholipid), while the minimal ratio was found in ampullary fluid collected near ovulation (1.5). Phospholipase activity was higher (P = 0.03) in isthmic oviductal fluid (20.4 +/- 3.2% product formed) than in ampullary oviductal fluid (14.6 +/- 1.4%); the lowest activity (12.6 +/- 1.7% product formed) was in fluid collected during the phase of the oestrous cycle immediately before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition and morphology of lipid droplets from oviduct epithelial cells.

This study was undertaken to determine the composition and morphology of lipid droplets in situ and isolated from oviductal epithelial cells and oviductal fluid. Oviductal epithelial cells were harvested enzymatically from oviducts of cows in either the luteal or the follicular stages of the ovarian cycle. Lipid droplets were isolated from cellular homogenates and characterized biochemically using thin layer chromatography. The morphology of lipid droplets in oviductal epithelial cells and in fractions isolated by ultracentrifugation from cellular homogenates was examined by electron microscopy. Lipid droplets isolated from oviduct epithelial cells varied in composition with the ovarian cycle and the oviductal region. There was more total lipid in droplets isolated from cows in the luteal than follicular phase of the ovarian cycle. Most of this difference was due to large amounts of esterified cholesterol present in the samples from luteal-stage animals. The most esterified cholesterol was found in droplets isolated from the oviductal isthmus of luteal cows. Droplets similar in lipid composition to those isolated from epithelial cells were found in oviductal fluid. Four distinct types of lipid inclusions were evident in electron micrographs of oviductal epithelia and characterized as osmiophilic droplets, lipofuscin-like clusters, lamellar structures, and composite bodies. All of the lipid inclusions were found in droplet isolates except for the extracted lipid portion of the composite body. The presence and diversity of oviduct epithelial lipid inclusions suggest that the oviductal epithelium may be very active in lipid metabolism, particularly cholesterol dynamics.

Synthesis and secretion of lipids by bovine oviduct mucosal explants.

The objectives of this study were to determine the types of lipid synthesized and secreted by the bovine oviduct, and to determine whether lipid synthesis and secretion varied with stage of the ovarian cycle and oviductal region. Oviduct explant cultures were prepared from cows killed during either the follicular or luteal stage of the oestrous cycle. Both stage of ovarian cycle and oviductal region affected lipid synthesis by oviductal explants in vitro. More lipid was synthesized by explants from follicular than from luteal-stage cows. Ampullar explants synthesized the greatest quantity of total lipid, followed by the preampulla and isthmus. Separation of extracted lipids from cultured tissue by high performance thin-layer chromatography (HPTLC) resolved phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, free cholesterol, free fatty acid, triglyceride and esterified cholesterol, all of which were synthesized during culture. The ampulla synthesized significantly more phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol than did the other regions. Culture supernatants from ampullary explants contained the most newly synthesized cholesterol when compared with other regions. The histochemical location of neutral lipid droplets in the epithelium of cultured explants paralleled the localization of radioactivity in autoradiographs of explant extracts. The results suggest that the oviduct synthesizes a variety of lipids, and that some of these are released into culture supernatants.

Neutral lipid droplets in bovine oviductal epithelium and lipid composition of epithelial cell homogenates.

This study was undertaken to locate and to quantify lipids within the oviduct epithelial cells that might serve as a source of luminal lipids. Oviduct epithelial cells were analyzed from 12 cows in either the follicular or luteal stage of the ovarian cycle. Histochemical differences in neutral lipid droplets and phospholipids containing choline were detected among the oviductal regions. Neutral lipid staining was greatest in the preampulla and the ampulla and least in the isthmus. Staining of phospholipids containing choline was similar in preampullary and ampullary epithelia but was concentrated in isthmic crypts. Neutral lipid droplets, detected fluorescently with Nile Red, were present in a greater percentage of preampullary epithelial cells (76.8 +/- 1.8) than ampullary (42.1 +/- 2.1) or isthmic cells (12.2 +/- 1.3). Neither ovarian cycle stage nor side of ovulation affected the concentration of epithelial lipid droplets. Colorimetric lipid assays determined that concentrations of free cholesterol and glycerides in the preampulla were higher than in other regions. Most esterified cholesterol was detected in the isthmus. These findings indicate that the bovine oviduct epithelium exhibits regional differences in phospholipid and neutral lipid distribution. Because many of the lipids studied affect gamete and early embryo membranes, localization differences may affect the functional variability of the oviductal regions.

Cation concentrations in fluid from the oviduct ampulla and isthmus of cows during the estrous cycle.

To detect variations in oviduct fluid cation concentrations, Ca++, Mg++, K+, and Na+ were determined for daily samples of blood serum and bovine oviduct fluid collected from indwelling isthmic and ampullary catheters. Isthmic oviduct fluid Ca++ concentration was significantly greater than that in ampullary fluid, particularly around estrus and ovulation. Maximum Ca++ concentrations found in isthmic oviduct fluid at estrus (2.57 +/- .22 mM) and at ovulation (2.50 +/- .29 mM) were similar to those of medium used for in vitro capacitation of bovine sperm. Concentrations of Mg++ in oviduct fluid differed significantly by estrous cycle stage, but not by oviduct region, and were consistently lower than those detected in serum. No relationships were found for K+ or Na+ with respect to region or stage, but K+ was generally higher in oviduct fluid than in serum. The concentration of K+ averaged over stage and region (4.46 +/- .13 mM) and the K+:Na+ ratio (.032 +/- .002) were similar to those reported in bovine in vitro capacitating and fertilizing media. Concentrations of Ca++ and Na+ from peritoneal fluid from nonstaged cows were similar to those of oviduct fluid or serum. The Mg++ concentration was greater, and K+ concentration was less, in peritoneal than in oviduct fluid.

Intraparenchymal striatal transplants required for maintenance of behavioral recovery in an animal model of Huntington's disease.

Rats which receive injections of kainic acid (KA) into the striatum show many of the anatomical, biochemical and behavioral abnormalities seen in patients with Huntington's disease. Recently, it has been reported that fetal striatal transplants into the lesioned striatum could normalize the neurological and behavioral abnormalities produced by the KA lesion. The present study examined the issue of transplant integration in producing behavioral recovery. In one experiment, lesioned animals with transplants located within the lateral ventricle were compared against parenchymally transplanted rats. It was found that unless the ventricular transplant grew into the lesioned striatum there was no recovery. The second experiment demonstrated that electrolytic destruction of a successful fetal striatal transplant could reverse the transplant-induced behavioral recovery. These results suggest that the integrity of the transplant is important in maintaining behavioral recovery. A continuing functional interaction between the host brain and transplanted tissue may be a vital element in the success of the fetal striatal transplant.

The development of daytime rearing behavior in methylazoxymethanol-treated rats: methodological considerations.

The development of daytime rearing behavior was studied in the offspring of pregnant rats which received injections of methylaxymethanol acetate (MAM) or saline during the 15th day of gestation. MAM and control rats were tested at 10, 15, 20, 25, and 30 days of age. The results indicated that the onset of rearing for both groups appeared at approximately 15 days of age, with no significant differences found between sexes. No rearing deficits were seen in MAM rats through 25 days of age despite the fact that these animals sustained greater than a 50% reduction in telencephalic mass. However, at 30 days of age MAM rats reared for significantly longer periods of time during each episode than did their control counterparts, although the actual number of rears did not differ between groups. The results are discussed in terms of neuroplastic events which follow MAM-induced damage and the need for multivariate research when analyzing rearing behavior.

The topography of amphetamine and scopolamine-induced hyperactivity: toward an activity print.

The increased locomotor activity induced by systemic injections of d-amphetamine or scopolamine in rats was studied in Digiscan Animal Activity Monitors. This multifactorial analysis of locomotion demonstrated that activity measures of horizontal (ambulatory), vertical (rearing), stereotypic, and rotational behaviors differed depending on dose and drug. The topographies of these activity variables may be unique for the dopaminergic and cholinergic systems underlying hyperactivity. These results are a first step toward a needed increase in the sophistication of behavioral pharmacological techniques, allowing for the development of specific activity prints for different classes of psychoactive agents.

Locomotor hyperactivity: effects of multiple striatal transplants in an animal model of Huntington's disease.

Huntington's disease is characterized by gross degeneration of the intrinsic neurons of the striatum, restless hyperkinetic choreiform movements and dementia. Rats which received injections of kainic acid have provided an extremely viable model for this extrapyramidal movement disorder. The present preliminary report investigated the effects of multiple homotopic transplantations of normal fetal Day 17 striatal ridge tissue into the lesioned striatum of male kainic acid-treated rats. Nine weeks after transplantation, the spontaneous nocturnal hyperkinetic locomotor abnormalities as measured by horizontal activity and total distance travelled were attenuated in the striatal transplanted animals compared to sciatic nerve transplanted controls. Similarly, the exacerbated response to d-amphetamine exhibited by the animal model was attenuated in the striatal transplanted animals. The striatal transplants reconstructed much of the gross morphology of the lesioned striatum, although acetylcholinesterase was found to be reduced.

Ontogeny of electrical activity of main olfactory bulb in freely-moving normal and malnourished rats.

Electroencephalographic activity (EEG) was recorded from the main olfactory bulb (MOB) in freely-moving, normally-nourished, (NP, normal-protein diet) and malnourished (LP, low-protein diet) rats from 4 days of age through adulthood. MOB EEG was analyzed for dominant frequency components using power spectral techniques. For NP rats, a single dominant frequency component (induced wave) was present in the MOB EEG at 4-6 days of age. From 10 days of age through adulthood, the MOB EEG contained two dominant frequency components (induced and intrinsic waves). Both the induced wave and intrinsic waves increased in center-frequency to reach maturity at approximately 30 days of age. Rats reared on low-protein diets (8% casein, prenatal and postnatal) displayed relatively permanent retardation in the development of induced wave center-frequencies and a delay in the development of the intrinsic wave center-frequencies. These results closely parallel the morphological development of the MOB in normally-nourished and malnourished rats.

Automated measurement of multivariate locomotor behavior in rodents.

Most automated methods of monitoring locomotion in animals have used mechanical equipment which measure only one dependent variable and are quite susceptible to a variety of experimental errors. Computer technology has advanced the mechanical means of measuring locomotor activity, allowing a new capability for simultaneously recording many aspects of locomotion. For example, the Digiscan System measures over 20 different indices of ambulatory, rearing, stereotypical and rotational behaviors over real time. Although standardization of some of these techniques is still needed, the computerized approach has already proven itself of value in neurobehavioral research. This paper describes the use of commercial systems in research.