A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs

A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55 degrees C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H(2)SO(4) catalysis for 1.5 h at 55 degrees C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.     

Influence of body condition and bovine somatotropin on estrous behavior, reproductive performance, and concentrations of serum somatotropin and plasma fatty acids in postpartum Brahman-influenced cows

Ninety-nine multiparous Brahman-influenced (1/4 to 3/8 Brahman) cows were managed to achieve low (BCS = 4.3 +/- 0.1; n = 50) or moderate (BCS = 6.1 +/- 0.1; n = 49) body condition (BC) to determine the influence of bovine somatotropin (bST) on estrous characteristics, reproductive performance, and concentrations of serum GH and plasma NEFA. Beginning 32 d postpartum, cows within each BC were assigned randomly to treatment with or without bST. Non-bST-treated cows received no treatment, and treated cows were administered bST (Posilac, 500 mg s.c.) on d -35, -21, and -7 before initiation of the breeding season. On d -7, all cows received an intravaginal, controlled internal drug-releasing (CIDR) device. On d 0 (initiation of the 70-d breeding season), the CIDR were removed and cows received prostaglandin F2alpha (PGF2alpha). Blood samples were collected from the median caudal vein of the cows at each bST treatment and at d -28 and 0. Estrous behavior was monitored by radiotelemetry during the first 30 d of the breeding season. Growth hormone was increased (P < 0.05) in low and moderate BC cows treated with bST. The percentage of cows detected in estrus during the first 30 d of the breeding season was decreased (P = 0.05) for low BC (64%) compared with moderate BC (82%) cows. The interval to first estrus tended (P = 0.07) to be shorter in low BC-bST-treated cows (3.7 +/- 1.9 d) than in moderate BC-bST-treated cows (9.6 +/- 1.8 d). During the first 30 d of the breeding season, cows in low BC had a decreased (P = 0.02) number of mounts received and increased (P = 0.001) quiescence between mounts compared with cows in moderate BC. The number of mounts received was reduced (P = 0.04) in bST-treated cows. More (P = 0.02) cows treated with bST became pregnant during the first 3 d of the breeding season compared with non-bST-treated cows. The cumulative first-service conception rate tended (P = 0.07) to be greater for bST-low BC cows than non-bST-treated cows in low or moderate BC. On d 0, NEFA were greater (P < 0.05) in bST-treated vs. non-bST-treated cows. Low BC and bST reduced the intensity of behavioral estrus in postpartum Brahman-influenced cows. However, bST increased the first-service conception rate during the first 30 d of breeding and pregnancy rates during the first 3 d of breeding in postpartum Brahman-influenced cows.     

Validation of candidate bovine reference genes for use with real-time PCR

Accurate quantification with real-time PCR requires the use of stable endogenous controls. Recently, there has been much debate concerning the stability of commonly used reference or housekeeping genes. To address this concern, a number of statistical approaches have been designed to analyse data and assist in determining the most appropriate reference genes for experimental comparisons. In this study, three programs, BestKeeper, Norm Finder, and geNorm were used to assess four candidate reference genes: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal protein large (RPLP0) and beta-actin, for use in expression profiling of individuals from divergent cattle genotypes subject to parasitic challenge with the cattle tick Boophilus microplus. Results demonstrated beta-actin and GAPDH were the most suitable reference genes in blood and could be used either individually or combined as an index to normalise data. RPLP0 was identified as the least stable gene, while 18S rRNA was omitted as being too highly expressed. As the recommendations on the most suitable reference genes varied between the programs, it is recommended that more than one should be utilised, to ensure the most robust experimental tools are selected.     

Evaluation of tropically adapted straightbred and crossbred cattle: postweaning gain and feed efficiency when finished in a temperate climate

Beef cows in the subtropical USA must be adapted to the stressors of the environment, typically supplied by using Brahman (Br) breeding. Calves produced in the region, however, are usually grown and finished in more temperate regions, and have a perceived reputation for poor ADG and feed efficiency during finishing. Compromised fertility and carcass quality often associated with the Br have increased interest in tropically adapted Bos taurus breed types. The objective of this study was to evaluate 3 breeds [An = Angus (Bos taurus, temperate); Br (B. indicus, tropical); and Ro = Romosinuano (B. taurus, tropical)] and all possible crosses during various segments of post-weaning growth, and for feed efficiency during the finishing phase. Steer calves (n = 473) born over 3 yr were weaned in late September, backgrounded for at least 21 d (BKG), shipped 2,025 km to El Reno, OK, in October, fed a preconditioning diet for 28 d (RCV), grazed wheat (Triticum aestivum L.) pasture from November to May (WHT), finished on a conventional feedlot diet (FIN), and serially harvested after approximately 95, 125, and 150 d on feed. Body weight and ADG during each segment were tested using a mixed model that included calf age at weaning, year (Y), breed of sire (SB), breed of dam (DB), and interactions. In addition, winter treatment (continuous wheat or reduced grazing of wheat with supplement) was included for the wheat and feedlot phases. Sire within SB × SB [and pen (barn × year) for feedlot phase] were considered random. The SB × DB interaction was significant for all traits (P < 0.01) except exit velocity taken at weaning and ADG during FIN, but both traits were affected by 3-way interactions with Y or harvest group. Tropically-adapted purebred steers had greater (P < 0.01) ADG than AnAn through weaning and BKG in FL but the reverse was true during the RCV and WHT segments. Similar, but less pronounced results were noted for F(1) steers with 100% tropical influence compared with those with only 50%. Heterosis was numerically greater for most traits for An × Br (11 to 64%) compared with An × Ro and Br × Ro (3 to 42%), which were similar. In a subset of the steers (n = 261), G:F was not influenced by level of tropical breeding, but tropically adapted steers were more efficient (P < 0.05) by residual feed intake. No heterosis was evident. These data show that in temperate zones, winter is the period when productivity of tropically adapted cattle is compromised.     

Fasciola hepatica: a light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wild-type flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica.     

Phase I study to determine the maximal tolerated dose and dose-limiting toxicities of orally administered idarubicin in dogs with lymphoma

Background

Idarubicin, a PO bioavailable anthracycline antibiotic‐class chemotherapeutic, could have substantial convenience advantages over currently available similar class agents in use that require IV delivery.

Objectives

The primary objective of this study was to determine the maximally tolerated dose (MTD), dose‐limiting toxicities (DLTs), and basic pharmacokinetic parameters of oral idarubicin exposure in dogs with lymphoma after a single oral dose. A secondary objective was to document preliminary antitumor efficacy in an expanded treatment cohort using the established MTD.

Animals

Client‐owned dogs with measurable lymphoma.

Methods

Dogs (n = 31) were enrolled in a prospective open label phase I study of oral idarubicin. By means of a 3 + 3 cohort design, dose escalations were made with 3 dogs per dose level, and the MTD was established based on the number of patients experiencing a DLT. Plasma concentrations of idarubicin and idarubicinol were determined by postdose sampling. Assessment of antitumor efficacy focused on evaluation of accessible, measurable lymph nodes and skin lesions by modified RECIST guidelines.

Results

The MTD in dogs > 15 kg body weight was 22 mg/m². Adverse hematologic events (neutropenia and thrombocytopenia) were the predominant DLT and generally correlated with higher plasma concentrations of idarubicin and idarubicinol.

Conclusions and Clinical Importance

PO administered idarubicin was generally well‐tolerated and had preliminary antitumor activity in dogs with lymphoma. Furthermore, the potential clinical advantage of a safe and efficacious oral anthracycline alternative supports further investigations of this agent in repeated‐dose, randomized clinical trials.