Lipid peroxidation is believed to play an important role in the pathogenesis of many diseases. Much research has therefore been devoted to peroxidation of different lipids in biomembranes and in model systems (liposomes) of different compositions. Yet, in spite of the relative simplicity of the liposomes, the existing literature is insufficient to reach definite conclusions regarding basic questions including the susceptibility of cholesterol to oxidation, its effect on the peroxidation of polyunsaturated phospholipids such as palmitoyllinoleoylphosphatidylcholine (PLPC) and how cholesterol influences the effect of water-soluble antioxidants such as urate on the peroxidation. The aim of the present study was to clarify these issues. Its major findings are that: (i) AAPH-induced peroxidation of cholesterol is slow and independent of the peroxidation of PLPC. In turn, AAPH-induced peroxidation of PLPC is not affected by cholesterol, independent of the presence of urate in the system. (ii) Cholesterol is not susceptible to copper-induced oxidation, but its inclusion in PLPC liposomes affects the peroxidation of PLPC, slowing down the initial stage of oxidation but promoting later stages. (iii) Addition of urate accelerates copper-induced peroxidation of PLPC in the absence of cholesterol, whereas in cholesterol-containing liposomes it inhibits PLPC oxidation. We attribute the complexity of the observed kinetics to the known cholesterol-induced rigidization of liquid crystalline bilayers.

Copper-induced peroxidation of liposomal palmitoyllinoleoyl-phosphatidylcholine (PLPC) is inhibited by alpha-tocopherol at micromolar concentrations. In our previous study we found that when the liposomes contain phosphatidylserine (PS), nanomolar concentrations of Toc were sufficient to inhibit peroxidation. In an attempt to gain understanding of the origin of this extreme antioxidative potency, we tested the antioxidative potency of 36 additional antioxidants and the dependence of their potency on the presence of PS in the liposomes. The results of these studies reveal that only 11 of the tested antioxidants possess similar antioxidative potency to that of Toc. These include trolox, butylated hydroxytoluene (BHT), curcumin, nordihydroguaiaretic acid (NDGA), diethylstilbestrol (DES), 2 of the 13 tested flavonoids (luteolin and 7,3',4'-trihydroxyflavone; T-414), alpha-naphthol, 1,5-, 1,6- and 1,7-dihydroxynaphthalenes (DHNs). Propyl gallate (PG), methyl syringate, rosmarinic acid, resveratrol, other flavonoids, as well as beta-naphthol, 1,2-, 1,3-, 1,4-, 2,3-, 2,6-, and 2,7-DHNs were either moderately antioxidative or pro-oxidative. For liposomes made of PLPC (250 mu M) and PS (25 mu M) the "lag" preceding copper-induced peroxidation (5 mu M copper) was doubled upon addition of 30-130 nM of the "super-active" antioxidants. We propose that the mechanism responsible for the extreme antioxidative potency against copper-induced peroxidation in PS-containing liposomes involves replenishment of the antioxidant in a ternary PS-copper-antioxidant complex. Based on structure-activity relationship of the 37 tested antioxidants, the "super-antioxidative potency" is attributed to the recycling of relatively stable semiquinone or semiquinone-like radicals. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

The study of gene function in vivo is considered one of the top achievements of modern biology, inasmuch as it provides tools to study gene function in the context of the whole animal. In chickens, techniques of DNA-mediated gene transfer are less advanced than in other animal or livestock models, and remain a significant challenge. The study presented here is the first to show that a hydrodynamics-based gene-transfer technique, originally developed for naked DNA transfer in mice, can be applied to chickens. Rapid injection of naked plasmids containing expression cassettes into the jugular vein of 6- to 10-day-old chicks resulted in specific expression of the transgenes. A CMV promoter-driven luciferase reporter gene was expressed at significant levels in the liver during the first 3 days post-injection with lower levels also detected in the kidney. Significantly, all injected birds showed detectable levels of luciferase expression. Similarly, injection of a plasmid containing the secreted human coagulation factor IX (hFIX) gene under the control of human alpha-1-anti-trypsin promoter resulted in detectable levels of the hFIX in the plasma during the first 2 days post-injection. The method described herein has the potential for a quick and simple route for gain and loss-of function experiments in chicken liver and kidney, as well as for studying systemic effects of secreted proteins and hormones.

The present study was conducted in an Assaf flock in which the FecB ( Booroola) mutation was segregated to determine whether the FecB mutation affects birthweight and the pre- and post-weaning growth rate of ewe lambs, as well as the mature bodyweight of ewes. Significant differences ( P = 0.01) in birthweight ( mean +/- s. e. m.) were found between BB ewe lambs (4.03 +/- 0.08 kg) and B+ and ++ ewe lambs (4.16 +/- 0.04 and 4.32 +/- 0.07 kg, respectively), which themselves did not differ significantly ( P > 0.05). An FecB-associated maternal effect on the birthweight of ewe lambs was also detected, with the birthweight of lambs born to BB mothers (3.93 +/- 0.08 kg) being significantly ( P < 0.0001) different from the birthweight of lambs born to B+ and ++ mothers (4.26 +/- 0.04 and 4.33 +/- 0.07 kg, respectively), which did not differ significantly. The genotypes of the lambs did not affect their preweaning growth rate. However, the post-weaning growth rate of ewe BB lambs ( 274 +/- 5 g day(-1)) was significantly ( P = 0.05) different from the similar ( P > 0.05) post-weaning growth rates of B+ and ++ lambs ( 284 +/- 3 and 290 +/- 4 g day(-1), respectively). The genotype at the FecB locus also affected the mature bodyweight of ewes, with that of BB ewes ( 67.3 +/- 1.4 kg) being significantly ( P < 0.001) different from the similar mature bodyweight of B+ and ++ ewes (70.8 +/- 1.1 and 70.1 +/- 1.7 kg, respectively).

BACKGROUND:Successful cryopreservation of a whole ovary may provide a solution for women with premature ovarian failure. The aim of this study was to evaluate the function of cryopreserved whole sheep ovaries both in vitro and in vivo.METHODS:Transplantation of frozen-thawed intact ovaries was performed on eight sheep by artery and vein anastomosis to the contralateral ovarian artery and vein. The remaining ovary was removed. Oocyte aspiration was performed 1 and 4 months post-transplantation. Serum progesterone levels were measured after 24 and 36 months. Magnetic resonance imaging (MRI) was carried out 12 months after transplantation.RESULTS:Progesterone activity was detected in three sheep from 24 to 36 months post-transplantation. Oocyte retrieval was successful in two sheep and parthenogenic activation has resulted in embryonic development up to the 8-cell stage. MRI revealed an intact ovary with small follicles and intact blood vessels.CONCLUSIONS:Whole ovaries, and the follicles and blood vessels they contain, are able to survive cryopreservation. In addition, MRI has shown that blood vessels were intact and that normal blood flow had resumed to the transplant. We conclude that immediate and long-term hormonal restoration and normal ovulation is possible after cryopreservation and transplantation of whole ovaries in sheep.

This is the first report of successful cryopreservation and transplantation of an intact ovary in a large animal (sheep). Three of eight transplanted sheep had resumed hormonal cyclicity.

Low fertility of cattle during the cool autumn is mostly associated with exposure to thermal stress in the previous summer. This study examined carry-over effects of summer heat stress and induction of enhanced follicular turnover, on the characteristics of preovulatory follicles during autumn. Holstein cows were treated with PGF2α and GnRH to induce six successive 9-day follicular waves during autumn. Intact cows served as controls. Follicular fluid concentrations of androstenedione were higher in treated cows, but those of IGF-I and progesterone did not differ between groups. The concentration of estradiol at the end of the treatment was higher, and the content per follicle increased with time and was numerically higher in treated cows. The volume of follicular fluid increased during the autumn and was greater in treated cows. A delayed effect of heat stress on preovulatory follicles was noted, and induction of follicular waves by GnRH was able to improve follicular characteristics in autumn.

RPCI-42 Bovine Bacterial Artificial Chromosome (BAC) library was screened with oligonucleotide sequences corresponding to specific elements of the 5 UTR sequence of bovine PC and to a region of the coding sequence. Two BACs that hybridized to all probes were selected for further analysis. A partial restriction map of the BACs was made with oligonucleotides corresponding to the coding region and the 89 and 110 bp elements of bovine PC 5 UTR. The BAC fragments that hybridized to the oligonucleotide probes were isolated and sequenced. The sizes of the cloned genomic PC 5 UTR fragments were verified by PCR, using genomic DNA from four cows. Sequencing data confirms the existence of a 178 bp exon that contains the 68 and 110 bp sequence elements of the 5 UTR for PC mRNA. The 178 bp exon appears to be the first transcribed exon in PC and the 68 and 110 bp 5 UTR sequences are most likely generated by alternative transcription start sites. Genomic sequence data also confirms that the 3 end of the 89 bp element is a discrete 41 bp exon. Regions within the genomic sequence adjacent to the 178 and 41 bp exons of the PC 5 UTR contain binding sites for TBP, Sp1, Ap1 and/or CEBP transcription factors. These data provide information about the arrangement of exons in the 5 UTR of PC and about putative promoter regions.

Novel object/ arena test was conducted on day 108 of gestation among sows housed in stalls (29 sows) and in pens with ESF (36 sows) to assess the fearfulness of sows when exposed to a novel arena and/or novel object as a measure of the housing-related stress experienced by the sows. A completely enclosed rectangular pen (4.78 m X 2.4 m) was divided into 10 equal segments and numbered one through 10. In the middle, opposite from the side of entrance a semicircle of radius 0.5 m was marked off (area A) for placement of the novel object, a fluorescent orange safety cone. The sows were moved into the pen, one at a time directly from their daily environment. The sow was observed for the first two minutes for the areas it entered within the arena. The sow was considered to be in an area if the snout entered the area. The novel object was placed in area A for the next three minutes and the sow was observed. Data on areas entered, time to approach the object, total time spent in the area A, time to first interaction with the object and number of interactions were recorded. The results of the test were compared using independent sample T-test and Kruskal-wallis ANOVA. More stall-housed sows (16) entered the area with novel object than sows housed in pens with ESF (13). Stall-housed sows took significantly less time (72.5 s) to enter the area with novel object than sows from pens with ESF (112.6 s). There was no significant difference among sows from both systems in terms of number of squares entered, time to have the first interaction and number of interactions with the novel object. The results were inconclusive as there was no possibility to account for the excitement in stall-housed sows when they were permitted to have a short walk and in a larger area during the test and the individual differences among sows.

BACKGROUND In lipid oxidation kinetics studies, prevalent cardiovascular disease has been associated with shortened lag phase, the length of time preceding the onset of oxidation. OBJECTIVES To examine, in vitro, copper-induced lipid oxidation kinetics in unfractionated serum from hemodialysis patients and to determine differences in kinetic parameters between patients with and without a history of CVD. METHODS Of the 76 patients enrolled in a study of oxidative stress in hemodialysis (44/76 with prevalent CVD, 53/76 males), 9 males with a history of myocardial infarction were selected and matched for age, diabetes and smoking status with 9 males from the non-CVD group. The kinetics of lipid oxidation was studied. Blood chemistry determinations including serum lipids, lipoproteins, hemostatic factors and serum malondialdehyde were obtained. Variables were compared using the t-test for independent samples with history of MI entered as the categorical variable. RESULTS Tmax, the oxidation kinetic parameter defined as the time at which the rate of absorbing product accumulation was maximal, was significantly shorter in dialysis patients with a history of MI than in those without (115.2 +/- 38.5 vs. 162.7 +/- 48.9 minutes, P = 0.04). Further, Tmax and MDA were negatively correlated to one another (r = -0.47, P = 0.04). Odds ratios indicate that each 1 minute increase in Tmax was associated with a 3% decrease in odds that a subject had a history of MI. CONCLUSIONS These findings indicate the presence of increased oxidative stress in hemodialysis patients with a history of MI.

Conception in dairy cows during autumn remains low even after summer temperatures decline. This is possibly a residual effect of heat stress on oocyte quality. Lactating Holsteins previously heat-stressed during summer were used in two experiments (n = 8 and 16 cows) in autumn to examine hormonal strategies for improving quality of oocytes. Follicles (3 to 8 mm in diameter) were aspirated by the ovum pick-up procedure on d 4 of two consecutive estrous cycles of treated and control cows. Oocytes were classified morphologically, matured in vitro, chemically activated, and cultured for 8 d. In Experiment 1, FSH (2 x 200 mg, at a 12-h interval) was injected on d 5 and 12 of cycle 1 (treated cycle). Before FSH, the percentage of good quality oocytes (grade I) and the cleavage rate averaged 52 and 24%, respectively, in both groups. During the FSH-treated cycle, more 6- to 9-mm follicles were observed. In the subsequent cycle, rates of grade I oocytes and cleavage were significantly higher in FSH-treated than in control cows (89 vs. 51% and 85 vs. 31%, respectively). In Experiment 2, recombinant bovine somatotropin (bST, 500 mg) was injected on d 4 and 18 of the treated cycle. The bST treatment increased the number of 3- to 5-mm follicles. Before bST, grade I (39%) and cleaved oocytes (40%), were similar across treatment groups. In the subsequent cycle, the percentage of grade I oocytes was significantly higher in treated than in control cows (72 vs. 26%), but cleavage rates were similar. Neither FSH or bST improved blastocyst formation, and regardless of treatment, few blastocysts were formed. Treatment with bST improved oocyte morphology, whereas FSH improved both oocyte morphology and cleavage rates during autumn following summer heat stress.

Selection for yield and the adoption of an intensive management system underpins the high milk production of the Improved Awassi and the Assaf dairy flocks in Israel. However, lamb production, which contributes some 40% of the income, remains relatively low at 1.2 and 1.6 lambs born/ewe lambing (LB/EL) for the Awassi and the Assaf, respectively. To increase the profitability, through improved lamb production, a breeding scheme was initiated in 1986 to introduce the B allele of the FecB (Booroola) gene to the Awassi and Assaf breeds. This led to the formation of the Afec–Awassi and Afec–Assaf strains with prolificacy of about 2.0 LB/EL. A marker assisted selection approach and the use of induced ovulation rate as selection criteria were incorporated into the breeding programs. The Awassi is the main type of sheep in the Middle East where about 80 million sheep of this breed and other related fat tail breeds are present. Distribution of the BB Afec semen or rams in those flocks, through regional breeding programs, can improve their productivity and their economics, facilitating the transition into a more intensive production system. The economic justification for launching such a breeding program depends mainly on the local lamb price.

The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.

Objective: Premature ovarian failure is a common complication of cancer patients treated by aggressive chemotherapy and radiotherapy. Cryopreservation of ovarian cortex slices, is proposed as a means of preserving future fertility. Currently however no pregnancy has been obtained, using frozen-thawed human ovarian cortex. One major obstacle is the ischemic damage post transplantation which results in loss of the majority of follicles. Xenotransplantation of human and animal ovarian cortex in immunosuppressed mice, has shown follicular loss and short term function of the graft due to ischemic damage. The purpose of this study is to develop a unique slow freezing technique that is suitable for whole organ cryopreservation. This would enable cryopreservation and vascular transplantation of an intact ovary. Design: Adult sheep oophorectomy with its blood vessels was performed. A slow freezing apparatus designed for whole ovary freezing was developed. Evaluation consists of granulosa cells (GC) viability staining and in-vitro fertilization of the retrieved oocytes. Retransplantation of the frozen-thawed ovary with reanastomosis of blood vessels was performed. Assessment of ovulation could be successfully performed by repeated laparoscopy. Next step consists of mating the sheep and obtaining a pregnancy. Materials/Methods: Institutional ethics approval was obtained. Eleven months old sheep underwent laparotomy under general anaesthesia. The ovaries were exposed with their vascular supply. Bilateral oophorectomy with vascular pedicles was performed. The ovarian artery was catheterized and cardioplegia solution was injected to perfuse the ovary. Then, a cryoprotectant solution consisting of UW solution containing 10% of DMSO was transfused for 5 minutes. In the preliminary experiment one ovary underwent slow freezing and the contralateral ovary served as a control. Slow freezing using IMT freezing device was performed as follows:the ovary was loaded into a 16 mm glass tube and seeding performed at −3°C or −6°C, then slow freezing at 0.1°C/min. to −7°C or −14°C. Thawing was performed at 90°C for 15 sec. followed by 38°C for 1 min. Oocytes and GC were retrieved from both ovaries, and oocytes were cultured and matured using in-vitro-maturation protocol. Following maturation, the oocytes were activated and grown to the blastocyst stage. In the next experiment, reanastomosis of the whole slow-frozen and thawed ovary was performed. Results: Oophorectomy with catheterization and resection of the ovarian artery and vein is feasible. Ovarian slow-freezing to −7°C or −14°C celsius could be performed over 55 minutes. Granulosa cells viability was 85% and 56% in the frozen whole ovary after freezing to −7°C and −14°C, respectively. We have retrieved 17 and 19 oocytes from the frozen whole ovary and the control ovary respectively. From the frozen whole ovary, 2/17 (12%) oocytes have reached the morula-blastocyst stage after in-vitro maturation and chemical activation. These results were lower than the results obtained from control ovary; 12/16 (75%). Additional data pertaining to ovulation and pregnancy of the transplanted frozen-thawed whole ovary will be presented at the meeting Conclusions: We have shown that vascular perfusion of the ovarian artery and slow freezing using a unique freezing apparatus enables preservation of the intact ovary and restoration of female fertility. Supported By: IMT LTD, Israel.

Insulin and glucose may be limiting factors for ovarian function in dairy cows genetically selected for high milk yield. The effects of nutrition on the intrafollicular content of insulin and glucose were investigated in Israeli Holstein dairy cattle fed a basic total mixed ration and producing 34-39kg of milk daily. In experiment 1, carried out in 11 oestrus-synchronised cows, little variation in insulin concentration was found in plasma sampled during the luteal phase, but high variation was found in plasma sampled during the follicular phase. Therefore, in order to prevent confounding the effects of diet and of phase in cycle in the following experiments, experimental diets were fed during the luteal phase of synchronised oestrus cycles. In experiment 2, designed as Latin-Square, six cows received sequentially diets containing 17.1 (control) or 19.7% of crude protein, using two sources of supplementary protein, i.e. soyabean meal (SBM) and corn gluten meal (CGM), differing in ruminal degradability and leucine content. When dry matter intake was used as covariant, plasma insulin on day 16 was 29.5 and 26.4% higher in cows fed diets containing SBM and CGM than in the control (P<0.05). In experiment 3, 17 cows were individually fed the basic diet and then switched to isoenergetic diets containing SBM (n=5), CGM (n=6) or corn grain (CG, n=6) given from day 10 to 16 of the synchronised oestrus cycle. On the eve of day 16, and in the morning of day 17, they were administered PGF(2alpha) and the content of 26 largest follicles was aspirated by using the transvaginal ovum pick-up technique. Follicles were sorted into two classes (preovulatory and subordinate) according to oestradiol concentration and the progesterone:oestradiol ratio in follicular fluid (FF). Higher concentrations of insulin (0.282 versus 0.127ng/ml, P<0.0001) and of glucose (0.614 versus 0.386g/l, P<0.002), were found in FF from preovulatory follicles. The insulin concentration in the FF of cows fed the CG diet was 26% higher than in their counterparts fed CGM (P<0.04), SBM being intermediate. Dietary effects did not reach significance in subordinate follicles. The finding that preovulatory follicular status is associated with increased intrafollicular insulin and glucose suggests that insulin is involved in follicular maturation. The nutritional effect on intrafollicular glucose and insulin may have practical implications to optimise feeding in dairy cows during phases of the oestrus cycle.

The effect of fat and bovine somatotropin (bST) on preovulatory follicular hormones and lipids was evaluated by feeding cows for 150 d from parturition a control diet, a control diet plus 0.55 kg/d of calcium soaps of fatty acids, or a control diet with 500mg of bST injected every 14 d. Fourteen days after a synchronized or natural estrus, cows were injected with a PGF2α analogue; 48h later, follicular fluid from all ovarian follicles >8mm was aspirated. Cows fed fat or injected with bST produced more milk and milk solids than did control cows, and cows on the bST treatment lost more body condition after calving than did cows on the other treatments. Both treatments changed the proportion of estradiol-active follicles (>400ng of estradiol/ml of follicular fluid) and the correlation between follicular fluid estradiol concentration and the total number large follicles per cow. In follicles aspirated between 60 and 90 DIM the percentage of estradiol-active follicles was 67, 40, and 0 for cows on the control, calcium soaps of fatty acids, and bST treatments, respectively. After 90 DIM, no differences existed between treatments in the percentage of estradiol-active follicles. Estradiol concentration in follicular fluid was correlated with DIM at follicle aspiration (r=0.51). The proportion of oleic acid in free fatty acids in plasma at 50 DIM was lower in control cows and was lower in follicular fluid of estradiol-active follicles. Both calcium soaps of fatty acids and bST had a considerable effect on follicular development and activity and the composition of fatty acids in follicles.

As a first step in evaluating the significance of our recently developed method of monitoring the kinetics of copper-induced oxidation in unfractionated serum, we recorded the kinetics of lipid oxidation in the sera of 62 hyperlipidemic patients and analyzed the correlation between oxidation and lipid composition of the sera [high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides]. We used six factors to characterize the kinetics of oxidation, namely, the maximal absorbance of oxidation products (ODmax), the maximal rate of their production (V max), and the time at which the rate was maximal (t max) at two wave-lengths (245 nm, where 7-ketocholesterol and conjugated dienic hydroperoxides absorb intensely, and 268 nm, where the absorbance is mostly due to dienals). The major conclusions of our analyses are that: (i) Both ODmax and V max correlate positively with the sum of concentrations of the major oxidizable lipids, cholesterol, and cholesteryl esters. (ii). The value of t max, which is a measure of the lag preceding oxidation and therefore reflects the resistance of the serum lipids to copper-induced oxidation, exhibits a negative correlation with HDL cholesterol. Although this finding accords with the observation of shorter lags for HDL than for LDL, it is apparently inconsistent with the role of HDL as an antirisk factor in coronary heart diseases.

More industrial and commercial value is placed on milk protein concentration but only a little is known about nutritional management of milk protein synthesis in dairy cows (DePeters and Cant, 1992). Some studies on this subject have shown a small positive effect of feed energy on milk protein concentration (Sutton, 1989). However, increase in milk production from the supply of dietary crude protein (CP) could be influenced by the supply and absorption from the small intestine of essential amino acids (AA), and this could be a nutritional betterment if it would protect from rumen fermentation process. Since dietary supply of AA some times gaves poor results it is still puzzling whether production is limited by specific AA or depends on bacterial and protozoon's proteins (BCP) supply to the small intestine. The objective of this study was to examine the effect of dietary CP levels and abomasal infusion of AA on milk yield and composition and to examine if these are key factors in regulating milk protein synthesis.

Four Holstein cows in midlactation were equipped with ruminal and abomasal cannulas and used to study the effect of synchronized degradation of crude protein (CP) and organic matter (OM) and feeding frequency on digestion and outflow of nutrients. A 4 x 4 Latin square design was used. Diets were arranged in a 2 x 2 factorial design; the four diets contained high ruminally degradable OM and high ruminally degradable CP, high ruminally degradable OM and low ruminally degradable CP, low ruminally degradable OM and high ruminally degradable CP, and low ruminally degradable OM and low ruminally degradable CP. In each period, cows were fed four times daily from d 1 to 14 and two times daily from d 15 to 28. Mean daily ruminal ammonia N concentration was reduced by high ruminally degradable OM, low ruminally degradable CP, and twice daily feeding. Fluctuation in ruminal ammonia N was lower when cows were fed four times daily than when cows were fed twice daily. Plasma urea N concentrations were lower for cows fed diets that were high in ruminally degradable CP. Higher CP flow in the abomasum was found for cows fed the diet containing high ruminally degradable OM and low ruminally degradable CP. Microbial dry matter and CP flow to the abomasum were higher for cows fed twice daily than for cows fed four times daily. Flow of OM in the abomasum was not altered by concentrations of ruminally degradable OM or CP. These results suggest that the available energy in the rumen (ruminally degradable OM) is the most limiting factor for ruminal N utilization under our experimental conditions. Use of these data may improve the prediction of plasma urea N.