Historical reconstruction of the population dynamics of southern right whales in the southwestern Atlantic Ocean.

Understanding the recovery of whale populations is critical for developing population-management and conservation strategies. The southern right whale (SRW) Eubalena australis was one of the baleen whale species that has experienced centuries of exploitation. We assess here for the first time the population dynamics of the SRW from the southwestern Atlantic Ocean at the regional level to measure numerically the effect of whaling and estimate the population trend and recovery level after depletion. We reconstructed the catch history of whaling for the period 1670-1973 by an extensive review of different literature sources and developed a Bayesian state-space model to estimate the demographic parameters. The population trajectory indicated that the pre-exploitation abundance was close to 58,000 individuals (median = 58,212; 95% CI = 33,329-100,920). The abundance dropped to its lowest abundance levels in the 1830s when fewer than 2,000 individuals remained. The current median population abundance was estimated at 4,742 whales (95% CI = 3,853-6,013), suggesting that the SRW population remains small relative to its pre-exploitation abundance (median depletion P2021 8.7%). We estimated that close to 36% of the SRW population visits the waters of the Península Valdés, the main breeding ground, every year. Our results provide insights into the severity of the whaling operation in the southwestern Atlantic along with the population´s response at low densities, thus contributing to understand the observed differences in population trends over the distributional range of the species worldwide.

Protection of the enzyme L-asparaginase during lyophilisation--a molecular modelling approach to predict required level of lyoprotectant.

Many novel therapeutic agents are proteins and peptides which need stabilisation due to their inherent instability in aqueous solution. Freeze-drying is an established method for protein stabilisation, although the use of additives is often necessary in order to preserve protein structure and activity during lyophilisation itself. The molecular interactions between protein and protective additive are as yet unclear. In this study, we examined the use of a range of saccharide additives to stabilise the model multi-subunit enzyme L-asparaginase during lyophilisation, assessed post-drying enzyme activity and quaternary structure, and related the extrapolated levels of additive necessary to provide full stabilisation to the theoretical levels predicted from an existing hypothesis using molecular modelling. It was found that each of the saccharides tested here displayed similar levels of protection towards L-asparaginase under the conditions used. Amounts of additive required to give full stabilisation to the enzyme were extrapolated from the activity data and were found to be in good agreement with theoretical amounts calculated from molecular modelling studies. Our data suggest that the existing hypothesis may be relevant to the prediction of optimum levels of lyoprotectant for the freeze-drying of proteins. However, further studies would be necessary in order to obtain a full picture of protein-additive interactions at the molecular level.

Optimizing the lyophilization cycle and the consequences of collapse on the pharmaceutical acceptability of Erwinia L-asparaginase.

The antileukemia enzyme, Erwinia L-asparaginase, occurs as a tetramer which can be dissociated by the stresses of lyophilization into four subunits (subunit M(r) 34 000 Da). Dissociation can be reduced by adding protectants to the formulation to stabilize the biopolymer, while the product should dry to form a pharmaceutically elegant, shelf-stable cake which is readily soluble. Using analytical ultracentrifugation, HPLC, and circular dichroism we have related structural dissociation of the enzyme during lyophilization to biological activity. Additives such as mannitol prevent ablation loss of vial contents and dry to form cosmetically elegant cakes but provide little biological protection, since during freezing they crystallize and are removed from the preparation. Excipients persisting throughout the cycle in the amorphous state provide improved biological protection, although high molecular weight compounds such as Dextran (M(r) 70000 Da) are most effective only during product freezing or storage. Low molecular weight sugars are protective throughout the cycle although formulations containing monosaccharides often exhibit low collapse temperatures (Tc) measured using a freeze-drying microscope or glass transition temperatures (Tg') measured by thermal analysis, but these formulations distort as drying progresses to form a collapsed, cosmetically unacceptable cake, with reduced activity, poor stability, a high moisture content, and reduced solubility. Collapse can be avoided by formulating with disaccharides, which display higher Tc temperatures than monosaccharides, or drying below Tc. Dried samples which persist in the amorphous state can also collapse when stored above their solid-state collapse temperatures when they decay at a faster rate than predicted by Arrhenius kinetics. The solid-state collapse temperature can be significantly decreased by the diffusion of moisture from the stopper into the dry product resulting in an increase in sample water content. Lyophilization cycle times can be reduced by analyzing collapse characteristics so that the relationship between product temperature and chamber pressure can be controlled so that drying rates can be optimized while ensuring that the product does not melt or collapse during sublimation.

Lyophilization of vaccines.

However effective a therapeutic agent may appear during clinical trials, its full potential as a marketable product can only be assured if the material can be stored and distributed in a stabilized form. This is particularly the case with labile biologlcals, such as attenuated vaccines, especially when these products need to be transported in tropical regions with limited distribution infrastructure.

Natural sodium sesquicarbonate fed for an entire lactation: influence on performance and acid-base status of dairy cows.

Forty-eight multiparous Holstein cows were blocked according to month of parturition, age, and previous milk yield and arranged in a randomized complete block design to evaluate the effect of a naturally occurring sodium sesquicarbonate on DMI, milk yield, milk composition, milk value, and systemic acid-base status. Cows were assigned at parturition to diets containing sorghum silage, alfalfa hay, concentrate, and 0 or 1% naturally occurring sodium sesquicarbonate (DM basis); cows were fed these diets for 308 d postpartum. Blood was collected every 4 wk via jugular venipuncture for analysis of pH, HCO3, partial pressure of O2, and partial pressure of CO2. Sesquicarbonate exhibited alkalogenic properties by increasing blood HCO3, partial pressure of CO2, and total CO2 for the 308-d lactation. Buffer tended to increase DMI and increased milk protein throughout lactation. During 0 to 56 d postpartum, sodium sesquicarbonate did not affect milk yield or composition. In midlactation (56 to 252 d postpartum), buffer increased milk protein content only. During 252 to 308 d postpartum, milk fat and protein contents increased with buffer supplementation. Hence, the value of milk yielded daily was similar for all cows. Effects of dietary buffer on all variables were more pronounced during late lactation.

Alpha 2-adrenoceptors in the guinea-pig uterus: heterogeneity in the circular and longitudinal smooth muscle layers.

Homogenate binding and functional studies have been undertaken to investigate the role of alpha 2-adrenoceptors on the circular and longitudinal myometrial layers of the dioestrous guinea-pig uterus. Each myometrial layer contained a single population of [3H]rauwolscine binding sites (KD values approximately 3 nM) for which yohimbine exhibited a higher affinity than prazosin, and xylazine a higher affinity than phenylephrine, indicating the presence of alpha 2-adrenoceptor binding sites. In circular myometrium, xylazine enhanced contractile responses, was more potent than either noradrenaline or phenylephrine in inhibiting forskolin-stimulated accumulation of cyclic AMP, and reduced the inhibitory effect of forskolin on phenylephrine-induced phosphatidyl inositol hydrolysis. In longitudinal myometrium xylazine enhanced contractile responses to phenylephrine but did not inhibit forskolin-stimulated accumulation of cyclic AMP. We conclude that alpha 2-adrenoceptor binding sites are present in both uterine layers and mediate uterine contractility possibly through different mechanisms.

Influence of dietary buffer value index on the ruminal milieu of lactating dairy cows fed sorghum silage and grain.

The objective of this study was to evaluate the influence of dietary buffer value index on ruminal fluid pH, buffering capacity, and buffer value index in lactating cows. Three Holstein cows averaging 18 +/- 10 DIM were used in a 3 x 3 Latin square with 3-wk experimental periods. Diets contained grain:sorghum silage DM ratios of 50:50, 60:40, and 70:30. By analysis, these diets had buffer value indexes of -74, -41, and -7. The analyzed dietary buffer value index of the total diets was higher than the index that was calculated from the buffer value index of individual ingredients. Although milk fat content tended to be highest for milk from cows fed the 50:50 concentrate to forage diet and although milk protein production was highest for cows fed the 70:30 concentrate to forage diet, milk yield, 4% FCM, milk fat yield, protein content, and milk fat content were not significantly affected by dietary buffer value index. Dietary buffer equivalents were calculated to be 11% of total buffering equivalents available to the cow, and dietary acid equivalents were 15% of total acid production in the rumen. Compared with ruminal acid production and salivary buffering in the rumen, dietary acid and dietary buffer contributions to the acid-base balance of the cow are minor quantitatively. Ruminal fluid pH, hydrogen ion concentration, buffer value index, buffering capacity, and total VFA were not affected significantly by dietary buffer value index. Hence, dietary acid-base status alone is inadequate as a predictor of the need for buffers in the diet of lactating cows.

Effects of inert fat on energy balance, plasma concentrations of hormones, and reproduction in dairy cows.

The objective was to determine the effects of dietary inert fat on estimated energy balance, hormones in plasma, and reproduction during early lactation. From wk 0 to 12 postpartum, 14 pluriparous Holstein cows were fed individually a TMR, and blood samples were taken twice weekly for quantification of IGF-I, progesterone, and cholesterol. During wk 5 to 12, one-half of the cows remained on the TMR, and the other half were fed the TMR containing inert fat at 1.8% of dietary DM. Estrous behavior was monitored twice daily, and body condition scores were recorded every 4 wk. Cows fed inert fat between wk 5 and 12 postpartum had similar concentrations of IGF-I in plasma but greater luteal phase progesterone secretions than cows fed the control diet. Total cholesterol in plasma also was greater in cows fed inert fat than in cows fed the control diet. Intervals to first, second, and third ovulation or estrus did not differ among cows fed control or inert fat diets. Body condition scores, daily DMI, and milk production were not affected by dietary inert fat. Inert fat fed to cows between wk 5 and 12 postpartum did not affect ovulatory activity but may enhance luteal activity.

Controlled ruminal infusion of sodium bicarbonate. 3. Influence of infusion dose on systemic acid-base status, minerals, and ruminal milieu.

Four ruminally cannulated, lactating Holstein cows were assigned to a 4 x 4 Latin square to monitor effects of intraruminal NaHCO3 infusion on temporal changes in ruminal and systemic acid-base status and mineral metabolism. Twice daily from 2 to 4 h postfeeding, cows were infused with 0, 110, 220, or 330 g of NaHCO3 dissolved in 3.8 L of water. All cows had access to their TMR of sorghum silage and concentrate (35: 65, DM basis) for 2 h twice daily. Ruminal fluid, blood, and urine were collected at feeding and every 30 min postfeeding for 12 h on the last day of each 14-d period. Total urine volume also was measured during this interval. Infusion of buffer increased ruminal fluid buffering capacity transiently at 4.5 h postfeeding but otherwise did not markedly affect ruminal fluid acid-base status. Systemic acid-base status was unaffected by the buffer primarily because renal excretion of base successfully reduced systemic base load. Urine volume increased in response to NaHCO3 infusion. Buffer infusion increased urinary excretion of Na, Mg, and K but decreased Ca excretion for 12 h postfeeding; Cl excretion was not affected. Buffer infusion tended to increase total VFA in ruminal fluid. Our data indicate that homeostatic mechanisms can eliminate exogenous base via the kidneys; hence, acid-base status was not perturbed by infusion of NaHCO3. The increased excretion of Mg and K with buffer infusion indicates that the dietary requirements for these minerals may be increased by NaHCO3. Although loss of Ca through the urine was reduced by buffer infusion, this reduction may indicate reduced availability of Ca to the cow. The diuresis accompanying large doses of NaHCO3 may increase dietary requirements for some minerals.

Some implications of structural collapse during freeze-drying using Erwinia caratovora L-asparaginase as a model.

When the enzyme Erwinia caratovora L-asparaginase was freeze-dried in mixtures of lactose and sodium chloride, biological activity and protein structure were preserved during drying. However, by altering the ratios of the excipients in the formulation it was possible to obtain products which were pharmaceutically acceptable or unacceptable as assessed by the criteria of dried cake appearance, moisture content or ease of reconstitution.

Influence of dietary fiber and buffer value index on the ruminal milieu of lactating dairy cows.

The influence of dietary buffer value index and dietary ADF content on ruminal fluid pH, buffering capacity, and buffer value index was measured. Four lactating Holstein cows (two primiparous) averaging 72 +/- 60 DIM were used in a 4 x 4 Latin square with 3-wk experimental periods. Treatments were a 2 x 2 factorial arrangement of TMR containing two ADF concentrations (16 and 21% of DM) and two buffer value indexes (calculated from analysis of individual dietary ingredients to be -200 and 0). Milk fat content and milk fat yield tended to be increased by high ADF, and protein yield tended to increase with low buffer value index and low ADF. Although the high ADF diets increased ruminal fluid pH, they reduced buffering capacity; because the magnitude of the pH increase was greater than the reduction in buffering capacity, ruminal fluid buffer value index was increased by added ADF. The high buffer value index diets reduced ruminal fluid pH and increased ruminal fluid buffering capacity; effects on pH outweighed those on buffering capacity so that the ruminal fluid index paradoxically decreased as the dietary index increased. Ruminal fluid acetate increased and propionate decreased as ADF increased. We conclude that ruminal fluid buffer value index increases with dietary ADF, likely because of reduced ruminal concentrations of fermentation acids. Because diets with the highest index produced the lowest ruminal indexes, dietary buffer value index must be studied further before it can be included in any model purporting to predict the need for supplemental dietary buffers.

Sodium bicarbonate or multielement buffer via diet or rumen: effects on performance and acid-base status of lactating cows.

Our objective was to compare the influence of dietary NaHCO3 and a multielement buffer on ruminal acid-base status and lactation performance of dairy cows. Five ruminally fistulated, primiparous and multiparous lactating Holstein cows averaging 123 +/- 21 d postpartum were assigned randomly to treatments in a 5 x 5 Latin square with 3-wk experimental periods. Treatments were a basal diet without supplemental buffers, with 1.5% NaHCO3 or 1.5% multielement buffer, or with NaHCO3 or multielement buffer solutions poured into the rumen via cannula at 2 h postfeeding. Addition of either buffer to the diet reduced ruminal fluid hydrogen ion concentration from 0 to 6 h postfeeding; only NaHCO3 reduced ruminal fluid acidity when dosed via the cannula. Addition of buffers via ruminal cannula appeared to retard the reduction in ruminal fluid acidity that normally occurs from 6 to 12 h postfeeding; this may have been related to a feedback mechanism inhibiting salivary buffer secretion. Buffering capacity of ruminal fluid tended to increase with buffer addition; the increase was greatest during infusion of NaHCO3. The ruminal fluid buffer value index increased by 4 units for control cows from early (0 to 6 h) to late (6 to 12) postfeeding; smaller increases were noted for addition of multielement buffer. This index was not different for NaHCO3 during these two intervals. Milk yield and DMI were not affected by buffer addition. Although milk fat content tended to be higher with the multielement buffer than with NaHCO3, it was not accompanied by the expected alterations in ruminal acid-base status. Therefore, this increase may be related to systemic effects of specific minerals in the multielement buffer rather than to a more stable ruminal environment. Based on the ruminal fluid buffer value index, NaHCO3 tended to maintain the most stable ruminal acid-base status.

Evaluation of a system for rating edema in dairy cattle.

Our objective was to evaluate a subjective edema rating system to quantitate edema severity. Edema was evaluated with 20 nulliparous Holstein heifers from 3 wk before their expected calving date until 3 wk postpartum. Severity of udder edema was evaluated independently daily by five people throughout the 68-d experiment; a 10-point rating system (0 = no edema, 10 = severe edema) was used. One of the people developing the scoring system served as the official scorer. The accuracy of the rating system, defined as the closeness of test evaluator scores to official scores, was highest for scores 2 to 8. The precision of the system, identified by the standard error of the mean, was similar for each score in the rating system. The largest disagreement between official and test evaluator scores was evident postpartum. The large number of defined points in this rating system and the similarity between official and test scores in this study indicate the usefulness of this tool to evaluate precisely the severity of edema in dairy cattle.

Influence of calcium chloride fed prepartum on severity of edema and lactational performance of dairy heifers.

Twenty Holstein heifers in a completely randomized design were used to evaluate the effects of prepartum dietary CaCl2 on the development and regression of edema and on subsequent lactational performance. Heifers were assigned to diets containing either 2.17% limestone or 1.5% CaCl2 (DM basis) at 3 wk before expected calving date. Except for the Ca sources, diets were identical. Test diets were only fed prepartum; at calving, all heifers were offered a lactation diet for 3 wk. Severity of edema was evaluated independently by five people daily throughout the experiment; a 10-point rating system was utilized to quantify the severity of edema. Calcium chloride reduced the severity of edema, although this response was most evident during the 1st wk that this salt was fed. The beneficial effects of CaCl2 on edema prepartum disappeared postpartum, when CaCl2 was removed from the diet. Indeed, edema was higher for heifers fed CaCl2 than for those fed limestone at 2 wk postpartum. Calcium chloride reduced DMI prepartum, but, following parturition, a compensatory increase in feed intake occurred. Postpartum daily DMI averaged 1.3 kg higher for heifers fed CaCl2 than for those fed the limestone diet prepartum. Milk yield and composition were somewhat lower for heifers fed CaCl2, although this response was most evident at 1 wk postpartum. Feeding CaCl2 prepartum to prevent milk fever also may reduce the severity of udder edema prepartum and increase feed intake during early lactation.

Influence of fiber form in a complete mixed ration on incidence of left displaced abomasum in postpartum dairy cows.

During the fall of 1982, increased incidence of left displaced abomasum (LDA) was seen in a group of cows fed a complete pelleted ration (3/16 inch) at the university's dairy center during the early postpartum period. Of these, 17.4% developed LDA when fed this experimental ration, whereas only 1.6% of the remaining cows in the herd developed LDA. The latter cows (as a control) were fed loose alfalfa hay, sorghum silage, and an 18% crude protein mix. Most of the fiber in the experimental diet or pelleted diet was alfalfa hay (30% as-fed basis) that was ground through a quarter-inch screen for incorporation into the pellet. Another 10% of the dietary fiber was contributed by cottonseed hulls. Thus, increased incidence of LDA may be associated with short length of the dietary fiber in the pelleted form.

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.

Influence of dietary cation-anion balance during the dry period on the occurrence of parturient paresis in cows fed excess calcium.

Our objective was to evaluate the effectiveness of a low dietary cation-anion balance (DCAB) in preventing milk fever and udder edema in dry cows consuming a high-Ca diet and to evaluate the effect of this diet on calves delivered by these cows. Seventy primiparous or multiparous cows and 50 pregnant heifers were offered alfalfa hay-based diets beginning 4 wk before their projected calving date. Diets contained 1.6% Ca and a DCAB of -3 or +9 mEq/100 g of diet DM. Blood and urine samples were collected weekly from 3 wk prepartum until 3 wk postpartum. Blood samples were collected from calves at parturition and weekly thereafter for 3 wk. Feeding a low vs high DCAB in a high-Ca diet for 3 wk prepartum did not reduce the incidence of milk fever; this lack of response may have been attributable to the relatively low DCAB of each diet and the small difference in DCAB between the two diets. Udder edema seemed to regress more rapidly postpartum for cows that had consumed the low DCAB during the dry period. Test diets fed to prepartum cows did not affect systemic acid-base status or plasma mineral content of their calves, although plasma Ca was somewhat lower for calves from cows consuming a low DCAB and was higher for calves from primigravid cows. Correlations of plasma mineral concentration of the cows with those of their calves were highest for plasma Ca (r = .75; P less than .001). We conclude that the prophylactic effects on the occurrence of milk fever of feeding a low DCAB during the dry period may be absent when diets contain greater than 1.6% Ca and DCAB is greater than or equal to -3. The cation-anion balance of the diet consumed by dry cows did not affect the acid-based status or plasma mineral content of their calves.

A buffer value index to evaluate effects of buffers on ruminal milieu in cows fed high or low concentrate, silage, or hay diets.

Our objective was to develop a buffer value index that would incorporate alterations in both ruminal fluid pH and buffering capacity as indicators of the influence of dietary buffering and alkalinizing agents on ruminal acid-base status. This index was evaluated using ruminal fluid from four lactating Holstein cows fed either sorghum silage or alfalfa hay in high or low concentrate diets. Ruminal fluid was incubated in vitro for 1, 2, 3, 4, or 5 h with no buffer or with 7.1 g of either NaHCO3, sodium sesquicarbonate, or a multielement buffer added per liter of ruminal fluid. Ruminal fluid pH was lower for diets based on high concentrate or alfalfa; buffering capacity between pH 5 and 7 was greater for high concentrate diets but was not affected by forage type. Ruminal fluid pH was higher for sesquicarbonate than for NaHCO3, the multielement buffer, or the control; however, ruminal fluid H+ concentration was similar between sesquicarbonate and NaHCO3, and both were lower than for the multielement buffer. Hydrogen ion concentration for the multielement buffer was lower than for the control. Buffering capacity was highest for NaHCO3, followed by sesquicarbonate, the multielement buffer, and the control. The buffer value index, which accounted for alterations in both H+ concentration and buffering capacity, was highest for NaCHO3, followed by sesquicarbonate, the multielement buffer, and the control. The poor response to the multielement buffer may be attributable to our relatively short incubation interval (less than 5 h). Dietary buffers increase both ruminal fluid pH and buffering capacity; both of these responses are beneficial.(ABSTRACT TRUNCATED AT 250 WORDS)

Controlled ruminal infusion of sodium bicarbonate. 2. Effects of dietary and infused buffer on ruminal milieu.

Four ruminally cannulated, lactating Holstein cows were assigned to a 4 X 4 Latin square to monitor the effects of NaHCO3 infusion on ruminal environment of cows receiving dietary sodium bicarbonate. Sodium bicarbonate (110 g) was mixed with 3.8 L of water and infused at a constant rate into the rumen from 0 to 2, 2 to 4, or 4 to 6 h postfeeding twice daily. All cows were fed sorghum silage and concentrate in a 35:65 DM ratio for 45 min twice daily. Ruminal fluid was collected at feeding and every 30 min postfeeding for 8 h on the last day of each 1-wk experimental period. Dry matter intake was not affected by NaHCO3 infusion. Yields of milk and its components were reduced with 4- to 6-h NaHCO3 infusion. At certain isolated times, especially during infusion, NaHCO3 infusion increased ruminal fluid buffer. Concentrations of ruminal fluid total VFA were not affected by NaHCO3 infusions, whereas acetate to propionate ratio tended to be reduced. Ruminal liquid volume tended to be increased by 0- to 2-h NaHCO3 infusion, and ruminal outflow rate tended to be reduced by the 2- to 4-h infusion. Intraruminal infusion of NaHCO3 into cows receiving supplemental dietary NaHCO3 altered ruminal acid-base status as typically reported for those receiving dietary NaHCO3; however, these alterations were not accompanied by shifts in ruminal VFA patterns or in milk composition that normally result from such feeding regimens. The effects of NaHCO3 infused directly into the rumen may be different from those of dietary NaHCO3 and are possibly related to the different time of entry into the rumen relative to feeding.