The steroidogenic capacity of several tissues has been shown to be regulated by lipoproteins. However, the ability of the various lipoprotein classes to affect steroid production in general and estradiol-synthesis in particular has not been established in bovine ovarian cells. In previous studies, we described alterations in the lipoprotein profiles in follicles of different sizes and corresponding changes in lipoprotein-receptor gene expression. In the present study, the effect of low-density lipoprotein (LDL)-enriched medium on aromatization competence of whole ovarian follicles was determined in vitro. Gene expression of aromatase increased and that of selective-uptake receptors decreased in the presence of LDL. These results suggest a role for LDL availability in folliculogenesis.

Objectives of the project: To evaluate the effects of a glucogenic supplement during the peripartum transition period on insulin, hepatic triglyceride accumulation, interval to first ovulation, and progesterone profile in dairy cows. To compare benefits of supplemental fats differing in fatty acid composition and fed prepartum on hepatic triglyceride accumulation, interval to first ovulation, progesterone profile, and uterine prostaglandin production in lactating dairy cows. To assess the differential and carry-over effects of glucogenic and fat supplements fed to peripartum dairy cows on steroidogenesis and fatty acids in ovarian follicles. To determine the carry-over effects of peripartum glucogenic or fat supplements on fertility in high producing dairy cows (modified in year 3 to Israel only). Added during year 3 of project: To assess the activity of genes related to hepatic lipid oxidation and gluconeogenesis following dietary supplementation (USA only). Background: High milk yields in dairy cattle are generally associated with poor reproductive performance. Low fertility results from negative energy balance (NEBAL) of early lactation that delays resumption of ovarian cycles and exerts other carryover effects. During NEBAL, ovulation of ovarian follicles is compromised by low availability of insulin and insulin-like growth factor-I (IGF-I), but fatty acid mobilization from body stores is augmented. Liver function during NEBAL is linked to the resumption of ovulation and fertility: 1) Accumulation of fatty acids by the liver and ketone production are associated with delayed first ovulation; 2) The liver is the main source of IGF-I. NEBAL will continue as a consequence of high milk yield, but dietary supplements are currently available to circumvent the effects on liver function. For this project, supplementation was begun prepartum prior to NEBAL in an effort to reduce detrimental effects on liver and ovarian function. Fats either high or low in unsaturated fatty acids were compared for their ability to reduce liver triglyceride accumulation. Secondarily, feeding specific fats during a period of high lipid turnover caused by NEBAL provides a novel approach for manipulating phospholipid pools in tissues including ovary and uterus. Increased insulin from propylene glycol (glucogenic) was anticipated to reduce lipolysis and increase IGF-I. The same supplements were utilized in both the USA and Israel, to compare effects across different diets and environments. Conclusions: High milk production and very good postpartum health was achieved by dietary supplementation. Peripartum PGLY supplementation had no significant effects on reproductive variables. Prepartum fat supplementation either did not improve metabolic profile and ovarian and uterine responses in early lactation (USA) or decreased intake when added to dry cow diets (Israel). Steroid production in ovarian follicles was greater in lactating dairy cows receiving supplemental fat (unsaturated), although in a field trail fertility to insemination was not improved.

Reduced reproductive performance of dairy cows during the summer is often associated with elevated temperature. Semen collected and cryopreserved during the summer may be of low quality and might contribute to the compromised fertility of dairy cows during this season. The present study examined the association between seasonality, semen quality and its potential to survive cryopreservation. A comparison between semen collected during the summer (July to August) and that collected during the winter (November to December) revealed the summer semen to be inferior, as reflected by low motility and high mortality of sperm. Furthermore, samples that were defined as good quality had changes in lipid concentration and fatty-acid composition in both the seminal plasma and cell compartment. In particular, semen collected during the summer had reduced levels of polyunsaturated arachidonic acid (20:4; P < 0.05) and decreased levels of linoleic acid (18:2; P < 0.05) in the cell compartment; corresponding reductions in cholesterol (P < 0.06) and fatty-acid concentrations (P < 0.001) were detected in seminal plasma of semen collected during the summer. In addition, we provided the first evidence for the existence of a very-low-density lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular lipids. Interestingly, the expression of VLDLr was three-fold greater in samples collected during the winter than in those collected in the summer (P < 0.01) and was negatively associated with saturated fatty-acid concentration (P < 0.018) but not with that of cholesterol. An opposite pattern was noted for samples obtained during the summer; mRNA expression of VLDLr was negatively associated with cholesterol concentration (P < 0.01) but not with that of saturated fatty acids. Such modifications associated with extracellular lipid utilization and fatty-acid composition might explain, in part, the reduced quality of summer semen.

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.

One of the key stimulators of intestinal development in the chick is physical exposure to feed, while feed withholding delays the onset of gut development. A delay of 24–72h in onset of feeding is quite common in the poultry industry due to variation in hatching time and hatchery treatments. As intestinal development occurs in concert with the development of the gut associated lymphoid tissue (GALT), we investigated the effects of short term feed withholding on development of GALT in broiler hatchlings. GALT activity was determined by antibody production (systemic and locally in the gut), distribution of B and T lymphocytes in the gut, expression of lymphocyte specific genes, and distribution of B and T lymphocytes in the cloacal bursa. Our findings show that while development of GALT in the foregut (duodenum, jejunum, ileum) was only slightly and temporarily impeded by feed withholding, GALT activity in the hindgut and the gut-related cloacal bursa was significantly delayed during the first 2 weeks of life: Systemic and intestinal antibody responses following rectal immunization to antigen were lower, colonization of the hindgut (cecum and colon) by T and B lymphocytes was delayed, as well as the expression of chIL-2 mRNA in hindgut T lymphocytes. We also found that the increase of B and T population size in the cloacal bursa was delayed with time. Full recovery occurred from 2 weeks of age. The 2-week vulnerable period should be seriously considered in circumstances where hatchlings are in transit for extended periods from hatcheries to farms.

The mucous layer that covers the intestinal absorptive surface acts as a barrier against bacterial translocation. The chicken gut contains a diverse bacterial population which interacts with the mucous layer. In this report, we studied the effect of changing the intestinal microbial populations on mucin dynamics by feeding 1-d-old chicks a control diet or that diet containing either antibiotic growth promoter (AGP) or a probiotic product for 14 d. Dietary AGP increased the proportions of Bifidobacterium species in the duodenum compared with the other groups. In AGP-fed chicks, the villous surface area was increased in the jejunum, goblet cell density was greater in the jejunum and ileum, and mucin glycoprotein levels in the duodenum were lower than in the other groups (P < 0.05). Feeding AGP increased the expression of mucin mRNA in the jejunum and ileum compared with controls. The dietary probiotic increased the proportion of Lactobacillus species in the ileum compared with the controls (P < 0.05) and significantly enlarged the goblet cell "cup" area throughout the small intestine compared with the other groups. Expression of mucin mRNA and the levels of mucin glycoprotein were greater in the jejunum of the probiotic-fed chicks compared with controls (P < 0.05). Neither the probiotic nor AGP treatments affected the thickness of the mucous adherent layer. These results indicate that both probiotic and AGP altered processes of mucin biosynthesis and/or degradation mediated via changes in the intestinal bacterial populations. These modifications in mucin dynamics influence gut function and health and may change nutrient uptake.

The intestinal lumen contains food and bacteria in close contact with the mucus layer, which lines the absorptive surface. This matrix contains IgA and other antibacterial substances and acts as a barrier to harmful components and organisms. The underlying epithelial layer is protected by intraepithelial lymphocytes at the junctions between enterocytes and by the action of IgA within the enterocytes. The luminal contents are continuously sampled by M cells and dendritic cells in lymphoid tissue, which then present antigens to the complete array of immune cells present in the lamina propria, which can then mount appropriate defenses.This system is immature at hatch with the intestinal lymphoid tissues developing in the first posthatch days, mainly in the distal intestines. Lymphocyte colonization begins in the first posthatch week, whereas functional T lymphocytes and the presence of IgA secreting cells are only observed in the second posthatch week, and maturation occurs later. Thus, the hatchling appears to be susceptible to pathogens in the immediate posthatch period.

Summer heat stress (HS) is a major factor in decreased reproductive performance in high-producing dairy cattle, possibly by affecting the steroidogenic capacity of ovarian follicles and ovarian follicular dynamics. In the present study, mRNA expression of cholesterol receptors was determined in bovine ovarian cells. Two endocytotic receptors (very-low-density lipoprotein receptor (VLDLr) and low-density lipoprotein receptor (LDLr)), and two selective-uptake receptors (scavenger receptor class B type 1 receptor (SRB1) and the lipoprotein-receptor-related protein 8 (LRP8)) were evaluated. Ovarian follicles in four diameter categories were evaluated from cows under non-heat stress (NHS) and HS conditions. As follicle size increased, expression of mRNA in NHS cows increased for the selective-uptake receptors, SRB1 and LRP8, and decreased (P<0.004) for the endocytotic receptors, LDLr and VLDLr. In contrast, in HS cows, mRNA expression did not significantly change (with increasing follicle diameter) for either receptor type. With increasing follicle diameter, cholesterol and fatty acid concentrations in the follicular fluid did not change in HS cows, whereas in NHS cows, cholesterol increased (P<0.008) and fatty acid decreased (P<0.0001). These changes paralleled those in the different lipoprotein fractions in the follicular fluid. In follicles from HS cows, the altered mRNA expression patterns for the endocytotic and selective-uptake receptors caused changes in the regulation of cholesterol supply at critical stages of folliculogenesis, which may play a role in the low turnover rates of ovarian follicles during the summer.

This study has used a direct approach to calculate the optimal dietary supply of amino acids for maintenance and growth in broiler chicks. Amino acid intake and accretion in the carcass in Ross 308 chicks was measured following the feeding of different experimental diets with controlled feed intake from hatching through 42 d. Increasing crude protein levels at constant amino acid:protein ratio improved growth and reducing the crude protein levels at constant amino acid:protein ratios depressed performance. Altering the protein at constant essential amino acid concentrations had little affect on growth. Plotting the amino acid intake per metabolic BW against amino acid accretion per unit of metabolic BW yielded a straight line whose slope represents the efficiency of utilization of the amino acid for growth; the intercept represents maintenance. These values were then used to calculate the amino acid intakes required for any BW and growth rate. For comparison, inputs were calculated for the growth targets recommended in the Ross manual and compared with the dietary recommendations. The measured results were similar to the overall empirical recommendations although values differed slightly, with threonine required at higher levels then recommended. Using the growth rates obtained in this trial, optimal amino acid supply was compared with the NRC (1994) recommendations. This indicated that optimal amino acid supply was higher then NRC (1994) recommendations for most amino acids. The calculation of optimal amino acid supply as a function of BW and growth will allow effective formulation of diets for maximal efficiency.

Apparent digestibility of crude protein (CP), lipid, carbohydrate, energy and fatty acids was measured in various feed ingredients fed to hybrid tilapia (Oreochromis niloticus×Oreochromis aureus) including fish and poultry meals, corn gluten, soybean meal, rapeseed meal, sunflower seed meal, wheat, corn, sorghum, barley and wheat bran. Chromic oxide was used as a non-absorbed marker. A diet compounded from a mixture of these ingredients was then used to examine the possibility of predicting the digestibility of formulated diets. In addition, the effect of pelleting or extrusion on digestibility of a compound diet was examined. Apparent digestibility of CP ranged from 75% to 97%, lipids from 72% to 90% and energy from 39% to 89% in the different ingredients. Apparent digestibility of carbohydrates was lower and ranged from 32% to 80%. Digestibility of fatty acids ranged from 75% to 90% with saturated fatty acids exhibiting digestibilities lower than unsaturated fatty acids. In a full-fat soy diet containing 19% fat, digestibility of lipids did not decrease. There were no significant differences in digestibility between a diet that was either pelleted or extruded. Tests conducted using a compound diet indicated that ingredient digestibility was additive for protein, lipids carbohydrates and energy. Diets for the hybrid tilapia may thus be formulated on the basis of digestibility of individual ingredients, for the nutrients examined in this study.

Ten pregnant heifers and 26 dry multiparous cows, 4 weeks before their expected parturitions, were blocked according to parity, BCS (body condition score) and BW (body weight) into two treatments: (1) control – cows fed until parturition with free choice of oat hay and 3kg DM (dry matter) per day of a lactating cow diet, and (2) treatment – cows were fed as the control plus 0.75kg (as fed, 860g/kg DM) of a supplement containing 410g/kg of non-structural carbohydrates (NSC) and 760g/kg total carbohydrates. Post-partum, both groups were fed the same lactation diet (168g/kg CP and 7.27MJL/kg DM). The mean live-weight of the treatment cows 2 days post-partum was 22.0 ± 9.2kg higher than control cows (P < 0.01). Mean daily milk and milk fat production during the first 120 days of the subsequent lactation were 38.5 and 36.9kg (P < 0.0003) and 33.1 and 30.4g/kg (P < 0.0004) for the treatment and control cows, respectively. Feeding a supplement containing a high proportion of NSC to dry cows reduced plasma glucose concentrations both pre-partum (P < 0.06) and post-partum (P < 0.01), and increased plasma insulin pre-partum (P < 0.04) and decreased it post-partum (P < 0.03) as compared with control cows. Pre-partum plasma β-hydroxybutyrate concentrations were enhanced (P < 0.04), and triglycerides reduced (P < 0.04), in the treatment cows with no post-partum differences. Pre-partum feeding with a supplement containing a high proportion of NSC can produce long term changes in metabolism with enhanced BW at parturition and increased milk and milk fat production through 120 days of the subsequent lactation.

The microflora in the gastrointestinal tract of broiler chickens influences digestion, health, and wellbeing. Analysis of chicken gut microflora has been mainly by culture-based methods. Studies using these techniques have been useful for identification and analysis of specific groups of bacteria, however, the use of enrichment medium precludes even relative quantitation of bacterial species. Recent advances in ribosomal DNA-based molecular techniques make it possible to identify different bacterial populations in environmental samples without cultivation. In this study, the intestinal microflora was examined using 16S ribosomal DNA (rDNA) targeted probes from bacterial DNA isolated from intestinal and cecal contents of chickens at 4, 14, and 25 d of age. The ribosomal gene sequence was amplified using PCR with universal primers to determine total bacterial DNA and specific primers directed at 6 bacterial species: Lactobacillus, Bifidobacterium, Salmonella, Campylobacter, Escherichia coli, and Clostridium. The use of universal primers extends these methods to allow determination of relative proportions of different bacterial species. The results indicated that in young chicks the major species present in the small intestines and ceca was Lactobacilli, with a Bifidobacteria population becoming more dominant in the ceca at older age. Clostridium was detected in some segments of the small intestine in young chicks. In older chickens, Salmonella, Campylobacter, and E. coli species were found in the ceca. This study has demonstrated the use of molecular techniques for determining relative proportions of bacterial species and monitoring pathogens in the chick gastrointestinal tract.

This study examined the microscopic structure of the small intestine and digestion and absorption in the hybrid tilapia, Oreochromis niloticus×Oreochromis aureus. The small intestine was lined on the luminal face with villi, which were narrower and more dense in the proximal small intestine and became wider and sparser distally. The columnar epithelial cells lining the villi were predominantly enterocytes containing a brush border, interspersed with mucus containing goblet cells approximately every 4–6 cells. These mucus-secreting cells contained both acid and basic mucins and increased in density in the distal and the terminal sections of the intestine. Proliferation of enterocytes was observed in the intervillus areas and lower third of the villus. Fish were fed diets of either fish meal or fish meal–wheat to which chromic oxide had been added as a non-absorbed reference standard. Activity of the pancreatic enzymes, lipase, trypsin and amylase were determined along the small intestine relative to the non-absorbed marker. Activity of these pancreatic enzymes was maximal in the proximal segments and decreased distally. Changes in the activity patterns of trypsin and amylase were observed with the different diets in the proximal small intestine. Net secretion of both N and fatty acids was found in the hepatic loop and absorption commenced distally decreasing in rate with distance from the pylorus. The pattern of N absorption in the proximal small intestine changed with the different diets. The structure and function of the small intestine of the tilapian omnivore thus are generally similar to mammals and digestion and absorption adapt with diet.

Reproductive failure associated with heat stress is a well-known phenomenon in avian species. Increased prolactin (PRL) levels in response to heat stress have been suggested as a mechanism involved in this reproductive malfunction. To test this hypothesis, laying female turkeys were subjected to 40 degrees C for 12 h during the photo-phase daily or maintained at 24-26 degrees C. Birds in each group received oral treatment with parachlorophenyalanine (PCPA; 50 mg/kg BW/day for 3 days), an inhibitor of serotonin (5-HT) biosynthesis, or immunized against vasoactive intestinal peptide (VIP). Both treatments are known to reduce circulating PRL levels. Nontreated birds were included as controls. In the control group, high ambient temperature terminated egg laying, induced ovarian regression, reduced plasma luteinizing hormone (LH) and ovarian steroids (progesterone, testosterone, estradiol) levels, and increased plasma PRL levels and the incidence of incubation behavior. Pretreatment with PCPA reduced (P < 0.05) heat stress-induced decline in egg production, increase in PRL levels, and expression of incubation behavior. Plasma LH and ovarian steroid levels of heat stressed birds were restored to that of controls by PCPA treatment. As in PCPA-treated birds, VIP immunoneutralization of heat-stressed turkeys reduced (P < 0.05) circulating PRL levels and prevented the expression of incubation behavior. But it did not restore the decline in LH, ovarian steroids, and egg production (P > 0.05). The present findings indicate that the detrimental effect of high temperature on reproductive performance may not be related to the elevated PRL levels in heat-stressed birds but to mechanism(s) that involve 5-HT neurotransmission and the induction of hyperthermia.

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed. Mol. Reprod. Dev. 68: 169–175, 2004. © 2004 Wiley‐Liss, Inc.