Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes.

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo.

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles

Zona pellucida-sperm binding assay for equine oocytes.

The binding of a spermatozoon to the zona pellucida is the first step in fertilization. The number of spermatozoa bound to a zona pellucida may reflect the functional status of both the oocyte and spermatozoa. The aim of the present study was to determine whether the stage of maturation of the equine oocyte affects the capacity of the zona pellucida to bind with spermatozoa. Cumulus-oocyte complexes (COCs) were collected from the ovaries of mares from abattoirs or were obtained in vivo by ultrasound-guided follicular aspiration. Oocytes were inseminated directly after collection or after 30 h of in vitro maturation (IVM) with semen from the same ejaculate. The results indicate that the immature equine oocytes collected from abattoirs and inseminated directly after collection bound fewer spermatozoa than did mature (metaphase I or II) oocytes inseminated after 30 h of IVM. A similar correlation was observed with the oocytes collected in vivo. The mean number of spermatozoa bound by mature oocytes collected from preovulatory follicles and inseminated directly after collection was 324 +/- 29, whereas the mean number of spermatozoa bound by immature oocytes from nonpreovulatory follicles inseminated without maturation was 222 +/- 26 (P < 0.05). This difference was not observed in the oocytes inseminated after maturation. It is postulated that immature equine oocytes bind fewer spermatozoa than do oocytes matured either in vivo or in vitro.

Influence of epidermal growth factor on in vitro maturation of equine oocytes.

The effect of epidermal growth factor (EGF) on the in vitro maturation rate of equine oocytes was examined. Oocytes were collected from an abattoir (Expt 1) or using ultrasound-guided follicular puncture in vivo (Expt 2). All oocytes with a compact or expanded cumulus at recovery were cultured for 30 h in: medium 1 (TCM199 + fetal calf serum (FCS) + crude equine gonadotrophin (CEG) + oestradiol + antibiotics); medium 2 (TCM199 + EGF); medium 3 (medium 1 without FCS + EGF); or medium 4 (medium 1 without CEG + EGF). In Expt 1, 84% (37/44) and 87% (40/46) cumulus expansion (P > 0.05), and 39% (22/57) and 9% (5/57) (P < 0.01) nuclear maturation, were observed in medium 1 and 2, respectively. In Expt 2, cumulus expansion was observed after culture in medium 1, 3 and 4 (30/30, 31/31 and 29/29, respectively). The nuclear maturation rate was significantly lower in medium 3 (6%, 2/36) than in medium 1 (43%, 16/37) (P < 0.01) and was higher in medium 4 (64%, 25/39) than in medium 1, although the effect was not significant (P = 0.07). In conclusion, 50 ng EGF ml(-1) alone was an effective substitute for crude equine gonadotrophin and the presence of EGF improves the nuclear maturation rate of equine oocytes.

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare.

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation.

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.

Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare.

The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro maturation and measured their maturation-promoting factor activity by histone H1 kinase assay. Puncturing once at the end of the follicular phase and once during the luteal phase, or three times during the follicular phase, yielded about 11 cumulus-oocyte complexes per 22 days. The maturation rate was different between the groups, 51% in group EF, 34% in group DL (p < 0.05), and 15% in group DF (p < 0.01), and it increased with an increase in follicular diameter (p < 0.05). After in vitro culture, the H1 kinase activity was lower in oocytes that remained in germinal vesicle or dense chromatin stages than in oocytes that reached metaphase I or metaphase II (p < 0.05). The H1 kinase activity was not different between oocytes in germinal vesicle stage after in vitro maturation and immature oocytes that were not cultured in vitro, and was higher in preovulatory oocytes that reached metaphase II in vivo than in the oocytes that reached metaphase II after in vitro maturation (p < 0.001). This is the first report on kinase activity in the equine oocyte.

Maturation-promoting factor (MPF) and mitogen activated protein kinase (MAPK) expression in relation to oocyte competence for in-vitro maturation in the mare.

In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We showed that MAPK and the two subunits of MPF were present in equine oocytes whatever the nuclear stage they reached after in-vitro culture and whatever the culture medium used. In incompetent oocytes, MAPK remained in its non-phosphorylated form, supposed to be inactive. In conclusion, the incompetence of equine oocytes to resume and complete meiosis is not due to the absence of p34cdc2, cyclin B or MAPK. Our results suggest that it is more probably due to a deficiency of regulators of MPF and/or to an inability to phosphorylate MAPK.

Chorionic gonadotropin secretion is associated with an inhibition of follicular growth and an improvement in oocyte competence for in vitro maturation in the mare.

This study reports the follicular growth and oocyte competence for in vitro maturation and fertilization under the influence of circulating eCG. Three to 7 successive ultrasound-guided follicular punctures were performed on 4 pregnant mares from Day 23 until Day 75 of pregnancy and on 5 control mares whose embryonic vesicle was crushed on Day 22. All follicles larger than 5 mm were punctured 24 h after the largest follicle reached 18 mm. Expanded cumulus oocyte complexes (COCs) were stained at recovery to analyze the nuclear stage. Compact COCs were cultured in vitro for 46 h and either stained or processed for in vitro fertilization (IVF) and stained 26 h after IVF. In the control group, no mares showed an increase in eCG levels, whereas all the pregnant mares had concentrations higher than 100 ng/ml from Day 37. The number of follicles flushed during each puncture attempt significantly decreased with time for 3 of 4 pregnant mares. No significant change in this number was observed for the 5 control mares. The maturation rate of the oocytes from follicles 10-14 mm was significantly higher in the pregnant vs. the control group (14 of 17, 82%, vs. 13 of 30, 43%). The difference was not significant for the oocytes from follicles smaller than 9 mm or larger than 15 mm. After IVF, no oocyte was fertilized. The results led us to conclude that eCG is associated with an inhibition of follicular growth and an improvement in oocyte competence for in vitro maturation.

A high-molecular-weight preovulatory stage-related protein in equine follicular fluid and granulosa cells.

The high-molecular-weight proteins of equine follicular fluid were examined to determine whether some polypeptides are unique to certain physiological conditions. Fluids from ovarian follicles of various diameters and physiological stages during the follicular phase were recovered by ultrasound-guided follicular aspiration. Granulosa cells and cumulus-oocyte complexes (COC) were recovered by scraping the intrafollicular wall during puncture. Follicular fluids and corresponding serum, as well as granulosa cell lysates, were analyzed by one-dimensional SDS-PAGE and silver staining. COC morphology was assessed microscopically. A 200-kDa protein band was demonstrated in fluids from preovulatory follicles, in natural conditions or after induction of ovulation. This protein band was absent in fluids from follicles at earlier stages, subordinate follicles, and serum. The presence of this protein at the preovulatory (PO) stage was ascertained through recovery of the fluid from follicles twice during their growth. Its appearance was time dependent after induction of ovulation but was not induced by an intrafollicular injection of a physiological dose of progesterone. We also demonstrated the presence of this 200-kDa protein in granulosa cells lysates recovered from preovulatory follicles. The expression of this protein in the follicular fluid was related to the cumulus aspect and chromatin configuration of the enclosed COC. No relation was found between its presence in the follicular fluid at the PO stage and subsequent ovulation of the punctured follicle or embryo production. The identification of this molecule is approached and discussed. These results show a novel PO stage-related protein in equine follicular fluid, which may be involved in the differentiation and maturation mechanisms occurring in the follicle during the preovulatory period.

Equine oocyte competence for nuclear and cytoplasmic in vitro maturation: effect of follicle size and hormonal environment.

Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle.

The ovine SOX2 gene: sequence, chromosomal localization and gonadal expression.

The SOX gene family consists of a large number of embryonically expressed genes capable of encoding putative transcription factors and related by a DNA-binding domain, the HMG-box. We cloned and characterized the ovine SOX2 transcript using the screening of a testis (12dpp) cDNA library with a probe containing the SRY-HMG-box and we performed 3'RACE experiments. The ovine SOX2 sequence is strongly conserved in comparison to the human, mouse and chicken homologues and is located on sheep chromosome 1q33. The SOX2 expression pattern in developing gonads is consistent with the hypothesis that this gene plays a role in the germ cell line.

A comparison of the biochemical composition of equine follicular fluid and serum at four different stages of the follicular cycle.

Samples of blood and follicular fluid were recovered from 27 Welsh Pony mares at 4 distinct stages of follicular development. Eighteen biochemical parameters were measured in each sample, including sodium, potassium, chloride, glucose, urea, creatinine, calcium, inorganic phosphate, total bilirubin, total protein, albumin, magnesium, triglyceride, total cholesterol, nonesterified fatty acids, alkaline phosphatase, gamma-glutamyltransferase and aspartate aminotransferase. The concentrations of progesterone, 17beta oestradiol and testosterone, pH and osmolarity, were also measured in all the follicular fluid samples. The concentrations of all proteins measured were lower in follicular fluid than serum whereas the reverse was true in the case of the lipids. Analysis of variance indicated that serum and follicular fluid concentrations of most of the parameters measured varied in parallel.

Follicular fluid is not a compulsory carrier of the oocyte at ovulation in the mare.

The aim of this study was to test the possibility that ovulation can occur from a preovulatory follicle emptied of its follicular fluid. Transport of the oocyte into the oviduct and fertilisation in 29% of cases demonstrated that ovulation can occur in the absence of follicular fluid but the higher fertility achieved in control mares (62.5%) suggested that follicular fluid does serve a role during ovulation, fertilisation and oviductal transport. Injection of horse oocytes into preovulatory follicles in mules after removal of the follicular fluid, followed by insemination of the mules with horse semen, resulted in the production of one horse x horse embryo.

Timing of in vivo maturation of equine preovulatory oocytes and competence for in vitro maturation of immature oocytes collected simultaneously.

The objects of this study were to monitor the development of the cumulus complex and nuclear maturation in oocytes recovered from preovulatory follicles following treatment to induce ovulation and to investigate the in vitro maturation competence of oocytes recovered from smaller nonpreovulatory follicles of varying size. All follicles > or =5 mm in pony mares were individually punctured at 0, 6, 12, 24 and 35 h after an injection of LH to induce ovulation. The recovery rates of oocytes were 64% from 55 preovulatory follicles, 22% from 32 subordinate follicles and 52% from 227 small follicles. Cumulus expansion of the preovulatory oocytes occurred at 12 h post LH treatment while the metaphase I and II components of nuclear maturation were not completed until 24 and 35 h post LH respectively. For nonpreovulatory follicles, the frequency of atresia and oocyte competence for in vitro nuclear maturation both increased with increasing follicular size.

Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare?

In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes to analyse the stages of nuclear maturation. The transfer technique did not impair significantly the final maturation of the recipient preovulatory follicle. The indigenous oocytes within the recipient follicles were recognisable by their larger expanded cumulus of yellow colouration due to high hyaluronic acid content; 7/12 of these oocytes were mature (metaphase II). Around half (42/86; 49%) of the oocytes transferred to preovulatory follicles were recovered subsequently. Most of them showed cumulus expansion (41/42, 6 of which were rich in hyaluronic acid), 13 (32%) were mature, 15 (36%) were immature and 13 (32%) were degenerate. When the indigenous oocyte of the recipient mare was mature, 38% of the transferred oocytes were mature, this rate being no different from the in vitro maturation rate of 46%. This study showed that in vivo maturation of immature oocytes by transfer into a preovulatory follicle in a recipient mare is possible. The maturation rate is not different from the in vitro maturation rate. The technique allows the generation of mature oocytes that have an expanded cumulus rich in hyaluronic acid, similar to the situation in preovulatory oocytes. This result has not been obtained in vitro previously.

Parentage testing of Day 10 equine embryos by amplified PCR analysis of microsatellites.

Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared to those of the dam to identify those of maternal origin. The paternal alleles were then searched for within the genotype of the 3 sires, and the stallion(s) that exhibited the particular allele was said to be compatible with the embryo for this microsatellite. In this way, the true sire was identified correctly for all 21 embryos.

Effect of short- and long-term treatments by a low level of dietary L-carnitine on parameters related to fatty acid oxidation in Wistar rat.

This study was designed to examine whether short- and long-term treatments by a low level of dietary L-carnitine are capable of altering enzyme activities related to fatty acid oxidation in normal Wistar rats. Under controlled feeding, ten days of treatment changed neither body weights nor liver and gastrocnemius weights, but succeeded in reducing the weight of peri-epididymal adipose tissues. Triacylglycerol contents were lowered in liver and ketone body concentrations were found slightly more elevated in blood. In the liver, mitochondrial carnitine palmitoyltransferase I (CPT I) exhibited a slightly higher specific activity and a lower sensitivity to malonyl-CoA inhibition, while peroxisomal fatty acid oxidizing system (PFAOS) was found to be less active. Carnitine supplied for one month reduced the mass of the periepididymal fat tissue, but not those of the other studied organs, and produced a slight but non-significant gain in body weight after ten days of treatment. In the liver, CPTI characteristics were comparable in control and treated groups, while PFAOS activity was less in rats receiving carnitine. Data show that L-carnitine at a low level in the diet exerted two paradoxical effects before and after ten days of treatment. Results are discussed in regard to fatty acid oxidation in mitochondria and peroxisomes, and to the possible altered acyl-CoA/acylcarnitine ratio with increased concentrations of L-carnitine in the liver.

Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats.

The effect of very low levels of dietary long-chain n-3 fatty acids on delta 6 desaturation of linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3), and on delta 5 desaturation of dihomo-gamma-linolenic acid (20:3n-6), in liver microsomes and its influence on tissue fatty acids were examined in obese and lean Zucker rats and in Wistar rats. Animals fed for 12 wk a balanced diet containing ca. 200 mg of long-chain polyunsaturated n-3 fatty acids per 100 g of diet were compared to those fed the same amount of alpha-linolenic acid. Low amounts of long-chain n-3 fatty acids greatly inhibited delta 6 desaturation of 18:2n-6 and delta 5 desaturation of 20:3n-6, while delta 6 desaturation of 18:3n-3 was not inhibited in Zucker rats and was even stimulated in Wistar rats. Inhibition of the biosynthesis of long-chain n-6 fatty acids was reflected in a decrease in arachidonic acid (20:4n-6) content of serum lipids when fasting, and also in the phospholipid fatty acids of liver microsomes. On the contrary, heart and kidney phospholipids did not develop any decrease in 20:4n-6 during fish oil ingestion. Docosahexaenoic acid (22:6n-3), present in the dietary fish oil, was increased in serum lipids and in liver microsome, heart, and kidney phospholipids.

Enhancement of activities relative to fatty acid oxidation in the liver of rats depleted of L-carnitine by D-carnitine and a gamma-butyrobetaine hydroxylase inhibitor.

This study was designed to examine whether the depletion of L-carnitine may induce compensatory mechanisms allowing higher fatty acid oxidative activities in liver, particularly with regard to mitochondrial carnitine palmitoyltransferase I activity and peroxisomal fatty acid oxidation. Wistar rats received D-carnitine for 2 days and 3-(2,2,2,-trimethylhydrazinium)propionate (mildronate), a noncompetitive inhibitor of gamma-butyrobetaine hydroxylase, for 10 days. They were starved for 20 hr before being sacrificed. A dramatic reduction in carnitine concentration was observed in heart, skeletal muscles and kidneys, and to a lesser extent, in liver. Triacylglycerol content was found to be significantly more elevated on a gram liver and whole liver basis as well as per mL of blood (but to a lesser extent), while similar concentrations of ketone bodies were found in the blood of D-carnitine/mildronate-treated and control rats. In liver mitochondria, the specific activities of acyl-CoA synthetase and carnitine palmitoyltransferase I were enhanced by the treatment, while peroxisomal fatty acid oxidation was higher per gram of tissue. It is suggested that there may be an enhancement of cellular acyl-CoA concentration, a signal leading to increased liver fatty acid oxidation in acute carnitine deficiency.