Zero-Order Release of Gossypol Improves Its Antifertility Effect and Reduces Its Side Effects Simultaneously.

Gossypol was considered a promising male contraceptive but finally failed due to two side effects: hypokalemia and the irreversibility of its contraceptive effect. Here we demonstrate that sustained zero-order release could be a solution for these problems. The in vitro release of gossypol from gossypol/PEG layer-by-layer films follows a perfect zero-order kinetics. In vivo tests indicate that the films can maintain the plasma drug concentration constant in male SD rats for ∼20 days for a 30-bilayer film. The plasma drug concentration is 2 orders of magnitude lower than the peak plasma drug concentration when administered orally and the daily dose is >50-fold lower than the commonly used contraceptive oral dose. However, significant antifertility effects were still observed. Furthermore, hypokalemia was not observed, and the antifertility effects can be reversed after a recovery period. The results suggest that zero-order release can significantly improve the desired antifertility effect of gossypol and, meanwhile, significantly reduce its side effects. We envision the drug could be developed to be an effective, safe, and reversible male contraceptive by zero-order release.

Metabolic Characterization of Dairy Cows Treated with Gossypol by Blood Biochemistry and Body Fluid Untargeted Metabolome Analyses.

To characterize the metabolic disorders of dairy cows treated with gossypol, 12 dairy cows were assigned to either a control group or a treatment group that was fed 1000 mg of gossypol per kilogram of dry matter feed for 28 days. Milk quality was adversely affected, as both milk protein and lactose levels were significantly decreased in the gossypol-treated group (3.40% vs 3.16%, P = 0.044; 5.15% vs 4.91%, P = 0.027; respectively). Plasma samples revealed increases in alanine aminotransferase (P = 0.092), choline esterase (P = 0.02), and glutathione transferase (P = 0.0005) and decreases in glucose (P = 0.076) in the gossypol-treated group. Mass spectrometry based comparative metabolomic analyses showed reduced concentrations of the gluconeogenesis precursor l-glutamine (P = 0.047), with significant decreases (P < 0.05) in plasma l-lysine, l-threonine, and homoserine levels after gossypol treatment. HDL-C and LDL-C levels in the gossypol-treated group were increased (P = 0.044) and decreased (P = 0.023), respectively. These results demonstrate that gossypol induced oxidative stress and hepatotoxicity; reduced peripheral lipid metabolism, and enhanced hepatic lipid accumulation; decreased amino acid bioavailability and milk protein synthesis; and decreased gluconeogenesis and milk lactose in dairy cows.

Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence.

1. The natural polyphenol gossypol possesses many therapeutic benefits. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro. 2. Metabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis. Clearance of gossypol was evaluated in bile duct cannulated rats and in the single-pass perfused rat intestine model. In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes. 3. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. In bile duct cannulated rats, considerable amounts of glucuronides (G1, G2 and G3; 58.8-83.2% of dose) and parent compound (5.0-20%) were excreted into bile after IV administration. In the perfused rat intestine model, gossypol was well absorbed with a [Formula: see text] (the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides (G1, G2 and G3) were excreted into the gut lumen (2.5%) and into the bile (4.8%). Biliary excretion of unchanged gossypol (6.0%) was comparable to that of glucuronides. Further, gossypol was subjected to rapid glucuronidation by liver and intestine microsomes. Reaction phenotyping showed that multiple UGT1A enzymes (including UGT1A1, 1A3, 1A7 and 1A8) are mainly responsible for gossypol metabolism. 4. In conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism.

Effect of expanded cottonseed meal on laying performance, egg quality, concentrations of free gossypol in tissue, serum and egg of laying hens.

Three hundred and sixty Hy-Line Brown hens, 40 week of age, were allocated to five treatments, each of which included four replicates of 18 hens. After an expanded process of cottonseed meal (CSM), free gossypol content in CSM was decreased from 1.24 to 0.40 g/kg. The dietary treatments were corn-soybean meal based diets including 6% CSM and 6%, 8% and 10% expanded cottonseed meal (ECSM). Hens fed 8% ECSM had higher (P<0.05) laying rate and average egg weight than those fed 6% CSM. The albumen height and Haugh unit in the control group, 6% and 8% ECSM groups were superior (P<0.05) to other treatments. Hens fed 6% CSM resulted in severe (P<0.05) egg yolk discoloration. Free gossypol (FG) concentrations in yolk and albumen and tissues of the 6% CSM group were greater (P<0.05) than those in any ECSM treatments. Hens fed 6% CSM and 10% ECSM had the highest (P<0.05) FG concentrations in the liver compared with those in the kidney and muscle, and higher (P<0.05) FG residues in yolk than those in albumen. In conclusion, FG in CSM can be reduced by 68% through an expanded process and ECSM can be available in laying hens at up to 10% of the total diet and an appropriate replacement of soybean meal with ECSM may improve performance in laying hens.

A phase I study of AT-101 with cisplatin and etoposide in patients with advanced solid tumors with an expanded cohort in extensive-stage small cell lung cancer.

BACKGROUND: A phase I, dose-escalation study of AT-101 with cisplatin and etoposide was conducted to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), safety and pharmacokinetics in patients with advanced solid tumors, with an expanded cohort in patients with extensive-stage small cell lung cancer (ES-SCLC) to assess preliminary activity. METHODS: In the dose escalation portion, increasing doses of AT-101 were administered orally BID on days 1-3 along with cisplatin on day 1 and etoposide on days 1-3 of a 21 day cycle. At the RP2D, an additional 7 patients with untreated ES-SCLC were enrolled. RESULTS: Twenty patients were enrolled in the dose-escalation cohort, and 7 patients with ES-SCLC were enrolled in the expanded cohort. The MTD/RP2D was established at AT-101 40 mg BID days 1-3 with cisplatin 60 mg/m2 and etoposide 120 mg/m2 on day 1 of a 21 day cycle with pegfilgrastim support. Two DLTs of neutropenic fever were seen at dose level 1. After the addition of pegfilgrastim, no additional DLTs were observed. Grade 3/4 treatment-related toxicities included: diarrhea, increased AST, neutropenia, hypophosphatemia, hyponatremia, myocardial infarction and pulmonary embolism. No apparent PK interactions were observed between the agents. Preliminary activity was observed with PRs in patients with ES-SCLC, high-grade neuroendocrine tumor, esophageal cancer and NSCLC. CONCLUSIONS: AT-101 with cisplatin and etoposide is well tolerated with growth factor support. Anti-tumor activity was observed in a variety of cancers including ES-SCLC, supporting further investigation with BH-3 mimetics in combination with standard chemotherapy for ES-SCLC.

Effect of replacing soybean meal protein with protein from upland cottonseed, Pima cottonseed, or extruded Pima cottonseed on production of lactating dairy cows.

Pima cotton production is increasing in the United States, but Pima cottonseed generally contains higher concentrations of the antinutritive pigment gossypol than conventional upland cottonseed. Heating promotes the reaction of gossypol with protein, reducing gossypol absorption and toxicity. The objective of this study was to assess the nutritional value for dairy cattle of Pima cottonseed cake (PCSC) that was heated and oil largely removed by an experimental extrusion process, compared with upland cottonseed (UCS) and Pima cottonseed (PCS). The PCS had greater crude protein (CP) and ether extract, less neutral detergent fiber (NDF) and acid detergent fiber (ADF), similar total gossypol, but higher (-)-gossypol isomer compared with UCS. Extrusion reduced lipid content by 73%, increased concentrations of CP, NDF, and ADF, and reduced total gossypol, (+)-gossypol, and (-)-gossypol in PCSC versus PCS. Forty lactating Holsteins (8 with ruminal cannulas) were blocked by days in milk into 5 squares in a replicated, incomplete 8 × 8 Latin square, and were fed diets containing, on a dry matter (DM) basis, 30% alfalfa silage, 31% corn silage, 21 to 25% high-moisture corn, and about 15% CP. Diets were fed as total mixed rations for ad libitum intake. Supplemental CP was from (1) solvent soybean meal (SSBM) only or 50% from SSBM plus 50% from (2) UCS, (3) PCS, (4) PCSC, (5) UCS plus PCS, and (6) UCS plus PCSC, or (7) 50% from expeller soybean meal (ESBM) plus 50% from PCS, and (8) 50% from ESBM plus 50% from PCSC. Periods were 4 wk long (total of 16 wk); production data were collected over the last 2 wk and blood and ruminal samples were taken on d 28 of each period. Data were analyzed using Proc Mixed of SAS (SAS Institute Inc., Cary, NC). Diet affected dry matter intake, with greatest intake on diet 6 and lowest intake on diets 1 and 3. The highest milk fat content was observed on diet 5 and the greatest fat yield on diet 7; fat content and yield were lowest on diet 1 (soybean meal control). Milk fat secretion was proportional to dietary fat content, indicating that cottonseed oil was used effectively for milk fat synthesis. We observed a trend for an effect on milk protein yield with the greatest protein secretion occurring on diet 7. Milk urea was lowest on diets 3, 7, and 8. Ruminal concentrations of branched-chain volatile fatty acids were lower, or tended to be lower, when PCSC replaced either UCS or PCS in the diet, suggesting reduced degradation and increased escape of PCSC protein. Among cottonseed-containing diets, total gossypol intake was lowest on PCSC, intermediate on PCS, and highest on UCS. Total gossypol and both (+)- and (-)-isomers of gossypol were higher in blood plasma on PCS and lower on PCSC than on the corresponding diets containing UCS, indicating that the extrusion process reduced gossypol absorption. In this trial, production on diets supplemented with UCS, PCS, or PCSC was comparable to that on diets containing soybean meal.

Effect of feeding of calcium hydroxide-treated or vitamin E-supplemented cottonseed meal on plasma gossypol levels, blood parameters, and performance of Bikaneri lambs.

To study the effect of feeding calcium hydroxide-treated or vitamin E-supplemented cottonseed meal (CSM) incorporated diets on plasma gossypol, blood parameters and animal performance, 24 male Bikaneri lambs of 6-7 months of age and of uniform body weight were divided into four groups of six animals each in a completely randomized design and respectively fed isonitrogenous and isocaloric concentrate mixtures containing 20% soybean meal (CON) or 40% raw CSM (RCSM), 40% raw CSM supplemented with 500 IU of vitamin E per head per day (ERCSM), and 40%, 1.5% calcium hydroxide-treated CSM (CaCSM) along with ad libitum wheat straw throughout 510 days of experimental feeding. The lambs on all the diets grew linearly throughout the experimental period. The total weight gain, in turn the average daily gain (ADG), was not affected by dietary variations. The daily intake of dry matter, crude protein (CP), digestible crude protein (DCP) and total digestible nutrients (TDN) were found comparable among lambs of all the groups. Though total gossypol intake was similar in RCSM, ECSM and CaCSM groups, however, free gossypol intake was significantly higher (P < 0.01) in RCSM, ECSM groups as compared to CaCSM group. Serum iron and blood hemoglobin levels were significantly (P < 0.05) lower in RCSM group as compared to CaCSM and CON groups, and ALT activity was significantly (P < 0.05) higher on RCSM group as compared to other groups. Plasma gossypol and osmotic fragility of erythrocytes were significantly (P < 0.05) increased in RCSM group as compared to CaCSM and ERCSM groups. However, there was no significant difference in the concentration of other blood/serum biochemical constituents among the lambs on different groups. Based on the results, it may be concluded that feeding of 40% CSM in the concentrate mixture of the diet in Bikaneri lambs did not have any major adverse effect in blood parameters and animal performance. Either calcium hydroxide treatment or vitamin E supplementation did not produce any major additional benefits.

Effects of different sources and levels of dietary gossypol on gossypol residues in plasma and milk of lactating cows.

Free gossypol residues in tissues or milk from feeding whole cottonseed and cottonseed meal were measured for their effect on health of dairy cows and humans. Forty lactating cows were randomly assigned to 5 treatments in a 60-d experiment to investigate the effects of sources and dietary level of gossypol on plasma and milk gossypol concentrations in lactating cows. Five experimental diets had identical net energy for lactation and crude protein content on a dry matter (DM) basis. Soybean meal was the main protein ingredient used in the control diet. Cottonseed meal (CSM) or whole cottonseed (WCS) substituted for part of the soybean meal in the other 4 diets. Gossypol levels in the 5 diets were 0 (control), 91.15 mg/kg of DM in CSM1, 117.31mg/kg of DM in CSM2, 385.43 mg/kg of DM in WCS1, and 611.13 mg/kg in WCS2. Yields of 3.5% fat-corrected milk were significantly higher for cows in the WCS2 group; cows in the CSM1 and WCS1 groups showed no differences but both were numerically higher than the control and CSM2 groups. Milk protein concentration was lower for cows consuming WCS1 compared with the control group. Lactose concentration was lower for cows in the CSM2 group compared with the WCS2 group, but no differences were observed among other diets. Aspartate aminotransferase in serum was significantly higher for the WCS2 group compared with the control and WCS1 groups, but no difference was observed with the CSM1 and CSM2 groups. Concentrations of gossypol in plasma and milk of cows in the WCS1 and WCS2 groups were both higher than those of the other groups. No adverse effects were observed on cows fed diets containing 12.0% CSM, and no gossypol was found in plasma and milk. When WCS comprised 15% of the diet DM, yields of 3.5% fat-corrected milk were increased in cows and gossypol was detected in plasma and milk but not at harmful levels.

Apogossypol derivatives as pan-active inhibitors of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins.

Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a series of 5,5' substituted apogossypol derivatives was synthesized that resulted in potent pan-active inhibitors of antiapoptotic Bcl-2 family proteins. Compound 8r inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 0.76, 0.32, 0.28, and 0.73 microM, respectively. The compound also potently inhibits cell growth of human lung cancer and BP3 human B-cell lymphoma cell lines with EC(50) values of 0.33 and 0.66 microM, respectively. Compound 8r shows little cytotoxicity against bax(-/-)bak(-/-) cells, indicating that it kills cancers cells via the intended mechanism. The compound also displays in vivo efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Together with its improved chemical, plasma, and microsomal stability relative to compound 2 (apogossypol), compound 8r represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.

Gossypol disrupts embryo development in heifers.

Our objectives were to determine the effects of dietary free gossypol (FG) intake on plasma and uterine gossypol concentrations and embryo development and viability before and after culture with gossypol. Fifty postpubertal Holstein heifers weighing (+/-SD) 406 +/- 34.5 kg at 11.5 mo of age were blocked by age and body weight (BW) and randomly assigned to 1 of 3 isocaloric and isonitrogenous diets differing in their FG content: control (0 mg of FG/kg of BW), moderate (17.8 mg of FG/ kg of BW), and high (36.8 mg of FG/kg of BW). Heifers were fed the diets for 70 d before superovulation and embryo collection. Superovulated heifers were flushed on d 5 after induction of ovulation, and early morulae were either stained, to determine the number and proportion of live and dead cells, or randomly assigned to an in vitro culture for 96 h in media containing either 0 or 10 mug/mL of gossypol acetic acid. Plasma and uterine gossypol concentrations increased with increasing gossypol intake. The number of low-quality embryos-ova was greater for the high than for the moderate and control diets. Embryos collected from the high diet had the least number of cells because of fewer live cells, and were smaller in diameter. Greater dietary gossypol reduced blastocyst development and extended the time to reach the blastocyst stage. Similarly, gossypol concentration at 10 microg/mL compromised in vitro development and increased the proportion of degenerated embryos at 96 h in culture. These findings provide in vivo and in vitro evidence that intake of 36.8 mg of FG/kg of BW per d and gossypol concentrations >7 microg/mL in plasma, in uterine flush, or in vitro compromise early embryo development, which might explain some of the negative effects of gossypol on the fertility of dairy cows.

Quantitative determination of apogossypol, a pro-apoptotic analog of gossypol, in mouse plasma using LC/MS/MS.

A simple and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method based on internal standard quantitation using apigenin as the internal standard has been developed and validated for the analysis of the gossypol analog apogossypol, a pro-apoptotic compound, in mouse plasma. The methodology involves protein precipitation of plasma samples followed by LC/MS/MS analysis. Ascorbic acid was added to the spiking solutions and plasma samples to stabilize the easily oxidized compound. Separation of apogossypol and the internal standard from the plasma matrix was achieved using a C18 column with a gradient elution profile consisting of 5mM ammonium acetate and methanol. The validated range of the method extended from 10 to 2000 ng/mL with accuracies of 85-115% and precision of <15%. The average recovery of apogossypol at three concentrations (50, 200 and 1000 ng/mL) assayed in triplicate using this methodology was determined to be 90.8+/-12.9%. Recovery for the internal standard (apigenin) at a concentration of 500 ng/mL was found to be 99.9+/-6.41%. Apogossypol concentrations of 50 ng/mL and above were found to be stable in extracted plasma for 24h when stored at 25 degrees C. This method has been applied to the determination of apogossypol concentrations in plasma collected from mice given an IV dose of apogossypol.

Effects of feeding whole cottonseed and cottonseed products on performance and carcass characteristics of finishing beef cattle.

Three experiments were conducted to determine the effects of whole cottonseed or cottonseed products on performance and carcass characteristics of beef cattle. In Exp. 1, 120 beef steers (initial BW = 381 +/- 31.7 kg) were fed steam-flaked corn-based finishing diets with 10% (DM basis) basal roughage, and whole cottonseed or individual cottonseed components (cottonseed hulls, meal, and oil). Over the entire feeding period, ADG did not differ (P = 0.95), but DMI increased (P = 0.07) and G:F decreased (P = 0.06) for steers fed the cottonseed diets compared with the control diet. Dressing percent (P = 0.02) and marbling scores (P = 0.02) of carcasses from steers fed the cottonseed diets were less than for steers fed the control diet. In Exp. 2, 150 beef steers (initial BW = 364 +/- 9.9 kg) were used to determine the effects of whole cottonseed or pelleted cottonseed (PCS) on performance and carcass characteristics. Cattle were fed steam-flaked corn-based finishing diets in which whole cottonseed or PCS replaced all of the dietary roughage, supplemental fat, and supplemental natural protein of the control diet. Over the entire feeding period, steers fed the cottonseed diets had lower (P = 0.04) DMI and greater (P < 0.01) G:F than steers fed the control diet. Carcass characteristics did not differ (P = 0.16 to 0.96) among dietary treatments. In Exp. 3, 150 beef heifers (initial BW = 331 +/- 17.1 kg) were used to determine the effects of PCS or delinted, whole cottonseed (DLCS) on performance and carcass characteristics. Heifers were fed rolled corn-based finishing diets in which cottonseed replaced the dietary roughage, supplemental fat, and all or part of the supplemental natural protein of the control diet. Over the entire feeding period, ADG, DMI, and G:F of heifers fed the control diet did not differ (P = 0.19 to 0.80) from those of the cottonseed diets; however, heifers fed the diets containing PCS had greater ADG (P = 0.03) and G:F (P = 0.09) than heifers fed diets containing DLCS. Carcass characteristics of heifers fed the control diet did not differ (P > or = 0.28) from those fed the cottonseed diets. Heifers fed the diets containing PCS had greater (P < or = 0.03) HCW, dressing percent, and LM area than those fed DLCS. Based on our results, whole cottonseed, or products derived from processing whole cottonseed, can replace feedstuffs commonly used in beef cattle finishing diets with no adverse effects on animal performance or carcass characteristics.

Effects of source of gossypol and supplemental iron on plasma gossypol in Holstein steers.

Four experiments were conducted to evaluate factors influencing concentrations of plasma total gossypol (TG) in 30 Holstein steers fed cottonseed products. At the end of each 28-d experiment, steers were weighed and blood samples were collected and analyzed for plasma TG concentrations. During the entire study, steers did not show any overt signs of gossypol toxicity. In the 28 d before experiment 1, 30 steers with a body weight (BW) of 273 kg were fed a standardization diet with 15.0% Upland whole cottonseed (WCS) that resulted in a mean intake of 9.08 g/d of TG per steer/d and a plasma TG of 1.66 microg/mL. In experiment 1, 30 steers were fed 1 of 5 diets with 15.0% Upland WCS, but different levels of supplemental Fe [0, 150, 300, 450, and 600 mg/kg of diet dry matter (DM)]. Average daily gain was not affected by level of Fe in the diet, but DM intake, plasma TG, and plasma TG response decreased linearly as Fe in diets increased. In experiment 2, steers were fed diets with 15.0% Upland cottonseed as whole, cracked, roasted, cracked-roasted, or extruded. Analysis of the seed revealed that roasting or extrusion markedly reduced free gossypol (FG) content. Minor effects on animal performance were observed, but plasma TG decreased with roasting or extrusion of seeds, with the greatest reduction when the seed was cracked and then roasted. In experiment 3, steers were fed 2 levels of WCS (7.0 or 14.0% of DM) with 3 levels of cottonseed meal (2.8, 5.5, or 8.5% of DM) in the diet. Animal performance was not altered by diet, but plasma gossypol concentrations and responses were greater in steers fed diets with more WCS, because of the greater FG intake. In experiment 4, 24 steers were fed diets with 15.0% cottonseed (Upland or Pima) either as whole or cracked. Pima cottonseed increased TG and FG intakes, which resulted in greater plasma TG concentration and response. Animal response to processing of cottonseed tended to differ according to type of cottonseed. However, feeding Pima and cracking of cottonseed increased gossypol availability and plasma TG concentrations.

The effects of varying gossypol intake from whole cottonseed and cottonseed meal on lactation and blood parameters in lactating dairy cows.

Effects of varying amounts of gossypol from whole Upland cottonseed (WCS) and cottonseed meal (CSM) were evaluated in 40 midlactation Holstein cows. After 14 d of pretreatment, cows were assigned to 1 of the 5 treatments for 84 d: control (no gossypol), 931 mg/kg total gossypol (TG) and 850 mg/kg free gossypol (FG) from WCS (moderate TG and high FG); 924 mg/kg TG and 91 mg/kg FG from CSM (moderate TG and low FG), 945 mg/kg TG and 479 mg/kg FG with equal amounts of TG from WCS and CSM (moderate TG and FG), or 1894 mg/kg TG and 960 mg/kg FG with equal amounts of TG from WCS and CSM (high TG and FG). Concentrations of plasma gossypol (PG) and its isomers were directly proportional to FG intake. Concentrations of PG reached a plateau after 28 d on treatment, and they were highest in cows receiving a diet with high TG and FG. Erythrocyte fragility differed among treatments and increased with increasing FG intake. Plasma gossypol returned to negligible concentrations 28 d after withdrawal of cottonseed products from the high TG and FG diet. Serum vitamin A was similar among treatments, but vitamin E increased with increasing FG intake. Serum enzymes were generally unaffected by treatments, but urea N increased in diets higher in TG and FG. Intake of dry matter was higher for the diet high in TG and FG than for the control diet, but was similar for other treatments. Cows receiving the high TG and FG diet produced more milk and 3.5% fat-corrected milk, with no changes in milk composition. Feeding a diet containing 1894 mg/kg TG and 960 mg/kg FG for 84 d increased PG concentrations and erythrocyte fragility and resulted in minor changes in blood metabolites and enzymes, but no detrimental effect on lactation performance was observed. Indicators of liver, kidney, and muscle cell viability suggest that the higher amounts of gossypol consumed in this study had only minor effects on those tissues in lactating dairy cows.

Effect of diets containing gossypol on blood parameters and spleen structure in tilapia, Oreochromis sp., reared in a recirculating system.

The high cost of fish meal in tilapia diets warrants the potential use of cottonseed meal (CSM) as an alternative source of high quality protein. The effects of varying levels of CSM (0, 25, 50, 75 and 100%) as fish meal protein replacement on growth, blood parameters, spleen characteristics, free and bound gossypol in blood plasma, haemoglobin and haematocrit were determined in tilapia. Gossypol (C(30)H(30)O(8)) is a polyphenolic substance found in cottonseed that has known toxic effects in fish. Tilapias (n = 219, average weight = 11.3 +/- 3.9 g) were randomly distributed into 15, 32-L glass aquaria, representing five dietary treatments and three replicates per treatment. Each aquarium containing 13-16 fish was supplied with thermoregulated, recirculating water (27 +/- 1 degrees C) at 1 L min(-1) flow rate and photoperiod was constant (12 h L/12 h D). Fish fed 25-50% CSM protein replacement showed similar body weights and total lengths as the controls at the completion of the 16-week trial. Fish fed 75 and 100% CSM protein replacement showed a significant decline in body weight and total length. Fish fed 25-100% CSM protein replacement had significantly lower haematocrit and haemoglobin (ANOVA/LSD, P < 0.05) compared with levels in controls. The decline was most prominent in groups fed diets with 50-100% CSM protein replacement. Total and free gossypol concentrations of blood plasma significantly increased with increasing levels of CSM replacement (P < 0.05). No gossypol was found in blood plasma of fish from the control group. The occurrence of immature and abnormal erythrocytes was significantly greater among fish fed 75 and 100% CSM diets compared with fish fed 0-50% CSM diets. Spleen-somatic index (spleen weight/body weight x 100) did not differ between control fish and fish fed 50-100% CSM diets. Spleen abnormalities, such as large depositions of haemosiderin and melanin pigments and proliferation of melano-macrophage centres, lymphocytic depletion of the white pulp areas (hypocellularity), and presence of vacuoles and necrotic areas were observed among fish fed 50-100% CSM protein diets. In general, the pathological effects of gossypol in tilapia (low haemoglobin and haematocrit levels, abundance of immature red blood cells or polychromatocytes, abnormal spleen morphology) were similar to the effects of vitamin E and/or vitamin C deficiencies observed in other studies.

Type of cottonseed and level of gossypol in diets of lactating dairy cows: plasma gossypol, health, and reproductive performance.

Objectives were to determine effects of altering gossypol intake by feeding whole linted Upland (WUP) or a 1:2 blend of WUP and cracked Pima (BUPCP) cottonseed on plasma gossypol (PG) concentrations, reproduction, and health of Holstein cows. Cows, 813, on three dairy farms were assigned to one of two diets starting at 13 +/- 11 d in milk for a 170-d experiment. Diets contained 717 and 951 mg of free gossypol/kg of dry matter from WUP and BUPCP, respectively. Concentrations of PG, as well as the proportion of total gossypol (TG) as the minus isomer were higher for cows fed BUPCP vs cows fed WUP. Conception rate at the first postpartum artificial insemination did not differ between treatments. However, cows consuming the higher gossypol diet had reduced subsequent conception rates and a lower pregnancy rate. Incidence of abortions increased in the higher gossypol diet, and cows that aborted or remained open had higher PG concentrations. Increasing PG concentrations resulted in reduced conception rates and extended days open. The probability of conception after the first artificial insemination declined at a decreasing rate as the plasma TG increased. Incidence of health disorders were unaffected by gossypol intake and PG concentrations. Similarly, gossypol intake and PG concentrations had no effect on culling or mortality. Six cows died in each diet, and none had postmortem signs compatible with gossypol toxicity. Consumption of a high gossypol diet for 170 d had no effect on health of lactating dairy cows, but it increased PG concentrations and impaired reproductive performance.

Increasing dietary levels of cracked pima cottonseed increase plasma gossypol but do not influence productive performance of lactating Holstein cows.

Lactating Holstein cows were fed diets with increasing levels of cracked Pima cottonseed to determine its effects on plasma gossypol concentrations as well as milk yield and composition and dry matter (DM) intake in a short-term study. All diets contained 12.8% cottonseed, 43.5% concentrate, and 43.7% chopped alfalfa hay on a DM basis. The proportion of whole Upland cottonseed to cracked Pima cottonseed in the four dietswas 100:0, 67:33, 33:67, and 0:100. Four primiparous cows were fed the diets in a 4 x 4 Latin square design, and three multiparous cows were fed the diets in a Youden square design with five periods. All periods were 35 d. Upland and cracked Pima cottonseed contained 0.64 and 1.00% total gossypol (DM) with 41 and 52% of gossypol as the (-) isomer, respectively. Gossypol is a natural defense compound in the plant that protects it against pests and diseases, but can have antinutritional quality effects when consumed by dairy cattle. Total plasma gossypol concentrations increased linearly with increasing proportions (100:0, 67:33, 33:67, and 0:100) of cracked Pima cottonseed in the diet for primiparous (4.4, 6.0, 7.7, and 8.9 microg/ml) and multiparous (4.3, 7.3, 9.7, and 11.4 microg/ml) cows, respectively. While primiparous cows responded similarly to gossypol intake, the response of plasma gossypol intake in multiparous cows differed among cows. This indicates the importance of animal variation when relating plasma gossypol levels with gossypol intake. Milk yield, as well as its components and DM intake, were not affected by increasing dietary inclusion levels of cracked Pima cottonseedup to 8.6% of DM intake for either primiparous or multiparous cows, even though plasma gossypol concentrations increased sharply over this dietary inclusion range. Although the highest dietary inclusion level of Pima cottonseed (i.e., 12.8%) numerically depressed performance of cows of both parities, these differences failed to reach statistical significance in these short-term trials with few cows.

Type of cottonseed and level of gossypol in diets of lactating dairy cows: effects on lactation performance and plasma gossypol.

Objectives were to determine the effects of feeding whole linted Upland (WUP) and cracked Pima cottonseed (BUPCP) to lactating cows on plasma gossypol concentrations and lactation performance. Holstein cows (n = 813) from three commercial dairies were assigned to one of two diets starting at 13+/-11 d in milk (DIM) for a 170-d experimental period. Cottonseed was included at 10% of the diet dry matter, and treatments consisted of replacing WUP with a 1:2 blend of BUPCP. Blood was collected from all cows at 61 and 91 DIM and from a subset of 56 cows on one dairy at 10, 33, 61, 91, 120, and 152 DIM. Intakes of free gossypol increased 32% in cows receiving the BUPCP diet, and concentrations of total gossypol (TG), as well as the proportion of TG as minus (-) isomer in plasma, were higher for cows fed BUPCP than WUP. Plasma TG increased linearly with increasing DIM, but cows fed the BUPCP, especially multiparous cows, had a higher increase in plasma TG over time. Yields of milk and milk components did not differ between the two treatments, although, yields of milk and milk components were greater in cows with higher plasma TG. Replacement of WUP cottonseed with BUPCP cottonseed increased plasma gossypol, but dry matter intake and yields of milk and milk components were not affected.

Gossypol and gossypolone enantiomers in tissues of rainbow trout fed low and high levels of dietary cottonseed meal.

Gossypol is an antifertilizing agent in males and females. However, gossypol and its metabolite, gossypolone, have also gained interest because of their anticarcinogenic activities. This paper examines for the first time both enantiomers of tissue gossypol and gossypolone in mature rainbow trout fed two diets containing low (15%) and high (60%) levels of cottonseed meal (CM) for 9 months. The gossypol concentration was highest in liver followed by kidney, intestine, testis, blood plasma, stomach, and muscle. Gossypol was detected in muscles of fish fed low- and high-CM diets (0.31 +/- 0.03 and 1.95 +/- 0.59 microg of total gossypol/g, wet basis, respectively). The (+)-gossypol enantiomer was predominantly retained in all tissues. The ratio of (-)- to total gossypol ranged from 30 to 44% in fish fed the high-CM diet and from 23 to 30% in fish fed the low-CM diet except for muscle tissue (44%). Higher gossypolone concentrations were found in intestine than in liver. Gossypolone, however, was not detected in blood plasma, muscle, and testis of fish fed the low-CM diet. The ratio of gossypolone to gossypol was highest in muscle (1.75), followed by intestine (1.59), stomach (1.50), kidney (0.43), liver (0.34), testis (0.28), and blood plasma (0.27). This study indicated that the retention of the (-)-gossypol enantiomer is dependent on dietary concentrations and that the oxidative conversion of gossypol to gossypolone occurs more actively in the digestive tract and muscle than in other tissues in rainbow trout.

Effects of gossypol from cottonseed on hematological responses and plasma alpha-tocopherol concentration of dairy cows.

The effects of feeding whole cottonseed (WCS) and bovine somatotropin (bST) administration on hematological responses and plasma alpha-tocopherol concentrations of lactating dairy cattle were examined. After parturition, multi and primiparous Holstein cows (n = 159) were assigned randomly to a 2 x 2 factorial arrangement of treatments consisting of (0 or 15% WCS and 0 or 208 mg of bST injected every 2 wk starting within 7 d after calving. Blood samples were collected from a subset group of 64 cows at 14, 28, 42, and 56 d postpartum. Blood was collected from all cows (n = 159) at 75, 96 and 120 d postpartum. Blood samples were analyzed for alpha-tocopherol and total and (+)- and (-)-gossypol in plasma. Erythrocyte osmotic fragility, hemoglobin and hematocrit also were determined in blood. The mean concentrations of alpha-Tocopherol, total, and (+)- and (-)-gossypol were higher in cows fed WCS regardless of bST administration and plateaued by d 75 postpartum. Hematocrit and hemoglobin concentrations were not affected by treatments. Erythrocyte osmotic fragility was higher in cows fed WCS, but the increase was attenuated when bST was injected (diet x bST interaction). No clinical signs of gossypol toxicity were observed in the cows consuming the WCS.