Parental Care, Destabilizing Selection, and the Evolution of Tetrapod Endothermy.

Parental care has evolved convergently an extraordinary number of times among tetrapods that reproduce terrestrially, suggesting strong positive selection for this behavior in the terrestrial environment. This review speculates that destabilizing selection on parental care, and especially embryo incubation, drove the convergent evolution of many tetrapod traits, including endothermy.

Anatomy, ontogeny, and evolution of the archosaurian respiratory system: A case study on Alligator mississippiensis and Struthio camelus.

The avian lung is highly specialized and is both functionally and morphologically distinct from that of their closest extant relatives, the crocodilians. It is highly partitioned, with a unidirectionally ventilated and immobilized gas-exchanging lung, and functionally decoupled, compliant, poorly vascularized ventilatory air-sacs. To understand the evolutionary history of the archosaurian respiratory system, it is essential to determine which anatomical characteristics are shared between birds and crocodilians and the role these shared traits play in their respective respiratory biology. To begin to address this larger question, we examined the anatomy of the lung and bronchial tree of 10 American alligators (Alligator mississippiensis) and 11 ostriches (Struthio camelus) across an ontogenetic series using traditional and micro-computed tomography (µCT), three-dimensional (3D) digital models, and morphometry. Intraspecific variation and left to right asymmetry were present in certain aspects of the bronchial tree of both taxa but was particularly evident in the cardiac (medial) region of the lungs of alligators and the caudal aspect of the bronchial tree in both species. The cross-sectional area of the primary bronchus at the level of the major secondary airways and cross-sectional area of ostia scaled either isometrically or negatively allometrically in alligators and isometrically or positively allometrically in ostriches with respect to body mass. Of 15 lung metrics, five were significantly different between the alligator and ostrich, suggesting that these aspects of the lung are more interspecifically plastic in archosaurs. One metric, the distances between the carina and each of the major secondary airways, had minimal intraspecific or ontogenetic variation in both alligators and ostriches, and thus may be a conserved trait in both taxa. In contrast to previous descriptions, the 3D digital models and CT scan data demonstrate that the pulmonary diverticula pneumatize the axial skeleton of the ostrich directly from the gas-exchanging pulmonary tissues instead of the air sacs. Global and specific comparisons between the bronchial topography of the alligator and ostrich reveal multiple possible homologies, suggesting that certain structural aspects of the bronchial tree are likely conserved across Archosauria, and may have been present in the ancestral archosaurian lung.

Linking structure and function in the vertebrate respiratory system: A tribute to August Krogh.

High rates of pulmonary gas exchange require three things: 1) that gases at the contact surface of the lung's capillaries are replenished rapidly from the environment; 2) that this surface is large and thin; 3) that the capillaries are effectively perfused with blood. In spite of this uniform requirement, lungs have evolved complex and highly diverse architectures, but we have a poor understanding of the drivers of this diversity. Here, I briefly discuss some of the diversity in gross anatomical features directing airflow in avian and non-avian reptiles. I also review new insights into the cellular anatomy of the blood-gas barrier, which in mammals is composed of specialized endothelial as well as epithelial cells.

Unidirectional pulmonary airflow patterns in the savannah monitor lizard.

The unidirectional airflow patterns in the lungs of birds have long been considered a unique and specialized trait associated with the oxygen demands of flying, their endothermic metabolism and unusual pulmonary architecture. However, the discovery of similar flow patterns in the lungs of crocodilians indicates that this character is probably ancestral for all archosaurs--the group that includes extant birds and crocodilians as well as their extinct relatives, such as pterosaurs and dinosaurs. Unidirectional flow in birds results from aerodynamic valves, rather than from sphincters or other physical mechanisms, and similar aerodynamic valves seem to be present in crocodilians. The anatomical and developmental similarities in the primary and secondary bronchi of birds and crocodilians suggest that these structures and airflow patterns may be homologous. The origin of this pattern is at least as old as the split between crocodilians and birds, which occurred in the Triassic period. Alternatively, this pattern of flow may be even older; this hypothesis can be tested by investigating patterns of airflow in members of the outgroup to birds and crocodilians, the Lepidosauromorpha (tuatara, lizards and snakes). Here we demonstrate region-specific unidirectional airflow in the lungs of the savannah monitor lizard (Varanus exanthematicus). The presence of unidirectional flow in the lungs of V. exanthematicus thus gives rise to two possible evolutionary scenarios: either unidirectional airflow evolved independently in archosaurs and monitor lizards, or these flow patterns are homologous in archosaurs and V. exanthematicus, having evolved only once in ancestral diapsids (the clade encompassing snakes, lizards, crocodilians and birds). If unidirectional airflow is plesiomorphic for Diapsida, this respiratory character can be reconstructed for extinct diapsids, and evolved in a small ectothermic tetrapod during the Palaeozoic era at least a hundred million years before the origin of birds.

New insight into the evolution of the vertebrate respiratory system and the discovery of unidirectional airflow in iguana lungs.

The generally accepted framework for the evolution of a key feature of the avian respiratory system, unidirectional airflow, is that it is an adaptation for efficiency of gas exchange and expanded aerobic capacities, and therefore it has historically been viewed as important to the ability of birds to fly and to maintain an endothermic metabolism. This pattern of flow has been presumed to arise from specific features of the respiratory system, such as an enclosed intrapulmonary bronchus and parabronchi. Here we show unidirectional airflow in the green iguana, a lizard with a strikingly different natural history from that of birds and lacking these anatomical features. This discovery indicates a paradigm shift is needed. The selective drivers of the trait, its date of origin, and the fundamental aerodynamic mechanisms by which unidirectional flow arises must be reassessed to be congruent with the natural history of this lineage. Unidirectional flow may serve functions other than expanded aerobic capacity; it may have been present in the ancestral diapsid; and it can occur in structurally simple lungs.

The Evolution of Unidirectional Pulmonary Airflow.

Conventional wisdom holds that the avian respiratory system is unique because air flows in the same direction through most of the gas-exchange tubules during both phases of ventilation. However, recent studies showing that unidirectional airflow also exists in crocodilians and lizards raise questions about the true phylogenetic distribution of unidirectional airflow, the selective drivers of the trait, the date of origin, and the functional consequences of this phenomenon. These discoveries suggest unidirectional flow was present in the common diapsid ancestor and are inconsistent with the traditional paradigm that unidirectional flow is an adaptation for supporting high rates of gas exchange. Instead, these discoveries suggest it may serve functions such as decreasing the work of breathing, decreasing evaporative respiratory water loss, reducing rates of heat loss, and facilitating crypsis. The divergence in the design of the respiratory system between unidirectionally ventilated lungs and tidally ventilated lungs, such as those found in mammals, is very old, with a minimum date for the divergence in the Permian Period. From this foundation, the avian and mammalian lineages evolved very different respiratory systems. I suggest the difference in design is due to the same selective pressure, expanded aerobic capacity, acting under different environmental conditions. High levels of atmospheric oxygen of the Permian Period relaxed selection for a thin blood-gas barrier and may have resulted in the homogeneous, broncho-alveolar design, whereas the reduced oxygen of the Mesozoic selected for a heterogeneous lung with an extremely thin blood-gas barrier. These differences in lung design may explain the puzzling pattern of ecomorphological diversification of Mesozoic mammals: all were small animals that did not occupy niches requiring a great aerobic capacity. The broncho-alveolar lung and the hypoxia of the Mesozoic may have restricted these mammals from exploiting niches of large body size, where cursorial locomotion can be advantageous, as well as other niches requiring great aerobic capacities, such as those using flapping flight. Furthermore, hypoxia may have exerted positive selection for a parasagittal posture, the diaphragm, and reduced erythrocyte size, innovations that enabled increased rates of ventilation and more rapid rates of diffusion in the lung.

Subglottal pressure and fundamental frequency control in contact calls of juvenile Alligator mississippiensis.

Vocalization is rare among non-avian reptiles, with the exception of the crocodilians, the sister taxon of birds. Crocodilians have a complex vocal repertoire. Their vocal and respiratory system is not well understood but appears to consist of a combination of features that are also found in the extremely vocal avian and mammalian taxa. Anatomical studies suggest that the alligator larynx is able to abduct and adduct the vocal folds, but not to elongate or shorten them, and is therefore lacking a key regulator of frequency, yet alligators can modulate fundamental frequency remarkably well. We investigated the morphological and physiological features of sound production in alligators. Vocal fold length scales isometrically across a wide range of alligator body sizes. The relationship between fundamental frequency and subglottal pressure is significant in some individuals at some isolated points, such as call onset and position of maximum fundamental frequency. The relationship is not consistent over large segments of the call. Fundamental frequency can change faster than expected by pressure changes alone, suggesting an active motor pattern controls frequency and is intrinsic to the larynx. We utilized a two-mass vocal fold model to test whether abduction and adduction could generate this motor pattern. The fine-tuned interplay between subglottal pressure and glottal adduction can achieve frequency modulations much larger than those resulting from subglottal pressure variations alone and of similar magnitude, as observed in alligator calls. We conclude that the alligator larynx represents a sound source with only two control parameters (subglottal pressure and vocal fold adduction) in contrast to the mammalian larynx in which three parameters can be altered to modulate frequency (subglottal pressure, vocal fold adduction and length/tension).

Virtual and augmented reality: New tools for visualizing, analyzing, and communicating complex morphology.

Virtual and augmented reality (VR/AR) are new technologies with the power to revolutionize the study of morphology. Modern imaging approaches such as computed tomography, laser scanning, and photogrammetry have opened up a new digital world, enabling researchers to share and analyze morphological data electronically and in great detail. Because this digital data exists on a computer screen, however, it can remain difficult to understand and unintuitive to interact with. VR/AR technologies bridge the analog-to-digital divide by presenting 3D data to users in a very similar way to how they would interact with actual anatomy, while also providing a more immersive experience and greater possibilities for exploration. This manuscript describes VR/AR hardware, software, and techniques, and is designed to give practicing morphologists and educators a primer on using these technologies in their research, pedagogy, and communication to a wide variety of audiences. We also include a series of case studies from the presentations and workshop given at the 2019 International Congress of Vertebrate Morphology, and suggest best practices for the use of VR/AR in comparative morphology.

Computational Fluid Dynamics Reveals a Unique Net Unidirectional Pattern of Pulmonary Airflow in the Savannah Monitor Lizard (Varanus exanthematicus).

This report models pulmonary airflow in the savannah monitor (Varanus exanthematicus) using computational fluid dynamics simulations, which are based on computed tomography data. Simulations were validated by visualizing the flow of aerosolized lipids in excised lungs with good but not perfect agreement. The lung of this lizard has numerous successive bronchi branching off a long intrapulmonary bronchus, which are interconnected by intercameral perforations. Unidirectional flow has been documented in the lateral secondary bronchi of the savannah monitor, but patterns of airflow in the rest of the lung remain unknown, hindering our understanding of the evolution of pulmonary patterns of airflow in tetrapods. These results indicate that the lung contains a unique net unidirectional flow, where the overall flow scheme is similar during expiration and late inspiration, but dissimilar during early inspiration. Air is transported net caudally through the intrapulmonary bronchus and net craniad through secondary bronchi, much like the pattern of flow in birds. The simulations show that many chambers feature flow in multiple directions during parts of the respiratory cycle, but some regions also show robust unidirectional airflow. Air moves craniad through secondary bronchi and between adjacent secondary bronchi through intercameral perforations. The first secondary bronchus, the hilar-cranial bronchus, contains tidal flow that may improve ventilation of the central and dorsal lung parenchyma. These results expand our understanding of flow patterns in varanid lungs and suggest lungs with net unidirectional flow as an evolutionary pathway between tidal flow and complete unidirectional flow in multicameral lungs. Anat Rec, 2020. © 2019 American Association for Anatomy Anat Rec, 303:1768-1791, 2020. © 2019 American Association for Anatomy.

Contribution of gular pumping to lung ventilation in monitor lizards.

A controversial hypothesis has proposed that lizards are subject to a speed-dependent axial constraint that prevents effective lung ventilation during moderate- and high-speed locomotion. This hypothesis has been challenged by results demonstrating that monitor lizards (genus Varanus) experience no axial constraint. Evidence presented here shows that, during locomotion, varanids use a positive pressure gular pump to assist lung ventilation. Disabling the gular pump reveals that the axial constraint is present in varanids but it is masked by gular pumping under normal conditions. These findings support the prediction that the axial constraint may be found in other tetrapods that breathe by costal aspiration and locomote with a lateral undulatory gait.

Lithophagy Prolongs Voluntary Dives in American alligators (Alligator mississippiensis).

Many vertebrates ingest stones, but the function of this behavior is not fully understood. We tested the hypothesis that lithophagy increases the duration of voluntary dives in juvenile American alligators (Alligator mississippiensis). After ingestion of granite stones equivalent to 2.5% of body weight, the average duration of dives increased by 88% and the maximum duration increased by 117%. These data are consistent with the hypothesis that gastroliths serve to increase specific gravity, and that the animals compensate by increasing lung volume, thereby diving with larger stores of pulmonary oxygen.

The right-to-left shunt of crocodilians serves digestion.

Abstract All amniotes except birds and mammals have the ability to shunt blood past the lungs, but the physiological function of this ability is poorly understood. We studied the role of the shunt in digestion in juvenile American alligators in the following ways. First, we characterized the shunt in fasting and postprandial animals and found that blood was shunted past the lungs during digestion. Second, we disabled the shunt by surgically sealing the left aortic orifice in one group of animals, and we performed a sham surgery in another. We then compared postprandial rates of gastric acid secretion at body temperatures of 19 degrees and 27 degrees C and rates of digestion of bone at 27 degrees C. Twelve hours after eating, maximal rates of gastric acid secretion when measured at 19 degrees and 27 degrees C were significantly less in the disabled group than in sham-operated animals. Twenty-four hours postprandial, a significant decrease was found at 27 degrees C but not at 19 degrees C. For the first half of digestion, dissolution of cortical bone was significantly slower in the disabled animals. These data suggest the right-to-left shunt serves to retain carbon dioxide in the body so that it can be used by the gastrointestinal system. We hypothesize that the foramen of Panizza functions to enrich with oxygen blood that is destined for the gastrointestinal system to power proton pumps and other energy-demanding processes of digestion and that the right-to-left shunt serves to provide carbon dioxide to gastrointestinal organs besides the stomach, such as the pancreas, spleen, upper small intestine, and liver.

Bone Microvasculature Tracks Red Blood Cell Size Diminution in Triassic Mammal and Dinosaur Forerunners.

Vertebrate red blood cells (RBCs) display a range of sizes, spanning orders of magnitude in volume in different clades [1]. The importance of this size variation to diffusion during exercise is reinforced by functional links between RBC and capillary diameters [2, 3]. Small RBCs, such as those of mammals (which lack nuclei) and birds, contribute to shorter diffusion distances and permit relatively fast O2 uptake kinetics [4]. Although constraints on RBC size have been tied to the cell's need to attend capillary sizes for effective gas diffusion [3], as well as to genome size evolution [5, 6], major questions persist concerning patterns of RBC size evolution and its paleobiological significance. Here, we evaluate the relationship between RBC sizes and bone histometry and use microstructural evidence to trace their evolution in a phylogeny of extinct tetrapods. We find that several fossilizable aspects of bone microstructure, including the sizes of vascular and lacunar (cellular) spaces, provide useful indicators of RBC size in tetrapods. We also show that Triassic non-mammalian cynodonts had reduced and densely packed vascular canals identical to those of some mammals and likely accommodated smaller, more mammal-like RBCs. Reduced channel diameters accommodating smaller RBCs predated the origin of crown mammals by as much as 70 million years. This discovery offers a new proxy for the physiologic status of the mammal and avian stem groups and contextualizes the independent origins of their increased activity metabolism.

On the origin of avian air sacs.

For many vertebrates the lung is the largest and lightest organ in the body cavity and for these reasons can greatly affect an organism's shape, density, and its distribution of mass; characters that are important to locomotion. In this paper non-respiratory functions of the lung are considered along with data on the respiratory capacities and gas exchange abilities of birds and crocodilians to infer the evolutionary history of the respiratory systems of dinosaurs, including birds. From a quadrupedal ancestry theropod dinosaurs evolved a bipedal posture. Bipedalism is an impressive balancing act, especially for tall animals with massive heads. During this transition selection for good balance and agility may have helped shape pulmonary morphology. Respiratory adaptations arising for bipedalism are suggested to include a reduction in costal ventilation and the use of cuirassal ventilation with a caudad expansion of the lung into the dorsal abdominal cavity. The evolution of volant animals from bipeds required yet again a major reorganization in body form. With this transition avian air sacs may have been favored because they enhanced balance and agility in flight. Finally, I propose that these hypotheses can be tested by examining the importance of the air sacs to balance and agility in extant animals and that these data will enhance our understanding of the evolution of the respiratory system in archosaurs.

Unidirectional pulmonary airflow in vertebrates: a review of structure, function, and evolution.

Mechanisms explaining unidirectional pulmonary airflow in birds, a condition where lung gases flow in a consistent direction during both inspiration and expiration in some parts of the lung, were suggested as early as the first part of the twentieth century and unidirectional pulmonary airflow has been discovered recently in crocodilians and squamates. Our knowledge of the functional anatomy, fluid dynamics, and significance of this trait is reviewed. The preponderance of the data indicates that unidirectional airflow is maintained by means of convective inertia in inspiratory and expiratory aerodynamic valves in birds. The study of flow patterns in non-avian reptiles is just beginning, but inspiratory aerodynamic valving likely also plays an important role in controlling flow direction in these lungs. Although highly efficient counter and cross-current blood-gas exchange arrangements are possible in lungs with unidirectional airflow, very few experiments have investigated blood-gas exchange mechanisms in the bird lung and blood-gas arrangements in the lungs of non-avian reptiles are completely unknown. The presence of unidirectional airflow in non-volant ectotherms voids the traditional hypothesis that this trait evolved to supply the high aerobic demands of flight and endothermy, and there is a need for new scenarios in our understanding of lung evolution. The potential value of unidirectional pulmonary airflow for allowing economic lung gas mixing, facilitating lung gas washout, and providing for adequate gas exchange during hypoxic conditions is discussed.

Similarity of Crocodilian and Avian Lungs Indicates Unidirectional Flow Is Ancestral for Archosaurs.

Patterns of airflow and pulmonary anatomy were studied in the American alligator (Alligator mississippiensis), the black caiman (Melanosuchus niger), the spectacled caiman (Caiman crocodilus), the dwarf crocodile (Osteolaemus tetraspis), the saltwater crocodile (Crocodylus porosus), the Nile crocodile (Crocodylus niloticus), and Morelet's crocodile (Crocodylus moreletii). In addition, anatomy was studied in the Orinoco crocodile (Crocodylus intermedius). Airflow was measured using heated thermistor flow meters and visualized by endoscopy during insufflation of aerosolized propolene glycol and glycerol. Computed tomography and gross dissection were used to visualize the anatomy. In all species studied a bird-like pattern of unidirectional flow was present, in which air flowed caudad in the cervical ventral bronchus and its branches during both lung inflation and deflation and craniad in dorsobronchi and their branches. Tubular pathways connected the secondary bronchi to each other and allowed air to flow from the dorsobronchi into the ventrobronchi. No evidence for anatomical valves was found, suggesting that aerodynamic valves cause the unidirectional flow. In vivo data from the American alligator showed that unidirectional flow is present during periods of breath-holding (apnea) and is powered by the beating heart, suggesting that this pattern of flow harnesses the heart as a pump for air. Unidirectional flow may also facilitate washout of stale gases from the lung, reducing the cost of breathing, respiratory evaporative water loss, heat loss through the heat of vaporization, and facilitating crypsis. The similarity in structure and function of the bird lung with pulmonary anatomy of this broad range of crocodilian species indicates that a similar morphology and pattern of unidirectional flow were present in the lungs of the common ancestor of crocodilians and birds. These data suggest a paradigm shift is needed in our understanding of the evolution of this character. Although conventional wisdom is that unidirectional flow is important for the high activity and basal metabolic rates for which birds are renowned, the widespread occurrence of this pattern of flow in crocodilians indicates otherwise. Furthermore, these results show that air sacs are not requisite for unidirectional flow, and therefore raise questions about the function of avian air sacs.

Impact of different wheat milling by-products in supplements on the forage use and performance of beef cattle consuming low-quality, tallgrass-prairie forage.

Two experiments were conducted to evaluate the impacts on forage use and beef cattle performance of incorporating divergent wheat milling by-products in a 30% CP supplement. The by-products were wheat bran (high fiber) and second clears (high starch). The by-products were added as 1) 100% wheat bran; 2) 67% wheat bran, 33% second clears; or 3) 33% wheat bran, 67% second clears to constitute approximately 47 to 49% of the supplement. In Exp. 1, 90 Hereford x Angus cows (BW = 554 kg) grazing winter, tallgrass-prairie range were fed the supplement treatments (2.27 kg/cow daily) from early December 1997 until calving (average calving date = 3/11/98). Cumulative BW and condition changes from trial initiation through calving were not significantly different among treatments. Similarly, significant treatment effects on cow pregnancy rates as well as calf birth weights, ADG, and ending weights were not evident. In Exp. 2, 16 ruminally fistulated Hereford x Angus steers (BW = 484 kg) were blocked by weight and assigned to one of the same three supplement treatments or to a negative control (forage only). Steers had ad libitum access to tallgrass-prairie hay (76.4% NDF, 3.1% CP) and were fed supplement at the same rate (relative to BW) as the cows in Exp. 1. Forage OM, NDF, and digestible OM intakes were lower (P < 0.01) for the negative control than for supplemented steers but were not significantly different among the supplemented steers. Digestion of OM was lower (P = 0.03) for the negative control than for supplemented steers, although significant treatment differences were not evident among the supplemented groups. Digestion of NDF was not affected (P = 0.49) by treatment. Within the context of the amount of supplemental protein offered, changes in the combination of wheat milling by-products in the supplement did not affect cow performance or intake and digestion of low-quality forage.

Effect of ruminal vs postruminal administration of degradable protein on utilization of low-quality forage by beef steers.

An experiment was designed to determine the effects of ruminal and postruminal infusions of ruminally degradable protein (casein) on intake and digestion of low-quality hay by beef steers. Twelve ruminally fistulated Angus x Hereford steers (initial BW = 563 kg) were blocked by weight and assigned to one of three treatments: control (C; hay only) or hay plus ruminal (R) or postruminal (P) infusion of 400 g/d of sodium caseinate. The trial consisted of five periods: 1) 10-d adaptation to the hay diet; 2) 7-d measurement of hay intake (without infusions); 3) 10-d adaptation to protein infusion treatments (intake measurements continued); 4) 7-d measurement of hay intake and digestibility (infusions continued); and 5) 3-d ruminal sampling period (infusions continued). Steers were given ad libitum access to tallgrass-prairie hay (3.4% CP, 76.6% NDF) throughout the study. Casein was administered once daily before feeding, either directly into the rumen or via anchored infusion lines into the abomasum. Hay intake was increased by supplementation (P < 0.01). Ruminal infusion elicited a greater (P = 0.04) increase in hay intake than postruminal infusion. Intake tended (P = 0.11) to be lower in period 4 than in period 2 for control steers but was greater in period 4 than in period 2 (P < or = 0.03) for both R and P steers. The increase in intake between periods 2 and 4 was greater for R than for P steers (P = 0.03). Supplementation improved diet OM digestion (P = 0.04) but not NDF digestion (P = 0.18); however, greater relative error for NDF digestion may have limited the ability to elucidate significant treatment effects. There were no differences in either OM digestion (P = 0.42) or NDF digestion (P = 0.35) between R and P steers. Plasma urea N at 0 and 3 h after feeding on the last day of the experiment was lower (P = 0.05) for C than for R and P steers, but no difference (P = 0.48) was evident between R and P steers. Ruminal ammonia N levels also were increased by supplementation (P < 0.01), with a much larger increase for R than for P steers (P < 0.01). Total VFA concentrations were not affected (P = 0.21) by treatment, but R steers exhibited lower proportions of acetate and higher proportions of isobutyrate, valerate, and isovalerate than P steers (P < 0.01). In conclusion, ruminal and postruminal infusion of a degradable protein source improved forage utilization, although the response in forage OM intake and total digestible OM intake was greater for ruminal infusion than for postruminal infusion.

Effect of supplementation frequency and supplemental urea level on dormant tallgrass-prairie hay intake and digestion by beef steers and prepartum performance of beef cows grazing dormant tallgrass-prairie.

Effect of supplementation frequency and supplemental urea level on forage use (Exp. 1) and performance (Exp. 2 and 3) of beef cattle consuming low-quality tallgrass-prairie were evaluated. For Exp. 1 and 2, a 2 x 2 factorial treatment structure was used, such that two supplements (30% CP) containing 0 or 30% of supplemental degradable intake protein (DIP) from urea were fed daily or on alternate days. In Exp. 1 and 2, supplement was fed at 0.41% BW daily or at 0.83% BW (DM basis) on alternate days. For Exp. 3, a 2 x 4 factorial treatment structure was used, such that four supplements (40% CP) containing 0, 15, 30, or 45% of supplemental DIP from urea were fed daily or 3 d/wk. Supplements were group-fed at 0.32% BW daily or at 0.73% BW (DM basis) 3 d/wk. In Exp. 1, 16 Angus x Hereford steers (initial BW = 252 kg) were blocked by BW and assigned to treatment. Urea level x supplementation frequency interactions were not evident for forage intake, digestion, or rate of passage. Forage OM intake (OMI) and total digestible OMI (TDOMI) were not significantly affected by treatment. Total-tract digestion of OM (P = 0.03) and NDF (P = 0.06) were greater for steers supplemented daily. In Exp. 2, 48 Angus x Hereford cows (initial BW = 490 kg) grazing winter tallgrass prairie were used. Significant frequency x urea interactions were not evident for BW and body condition (BC) change; similarly, the main effects were not substantive for these variables. In Exp. 3, 160 Angus x Hereford cows (initial BW = 525 kg) grazing dormant, tallgrass prairie were used. Supplement refusal occurred for cows fed the highest urea levels, particularly for cows fed the supplement with 45% of the DIP from urea 3 d/wk, and supplement refusal increased closer to calving. A frequency x urea interaction (P = 0.02) was observed for prepartum BW changes. As supplemental urea level increased, prepartum BW loss increased quadratically (P = 0.02); however, a greater magnitude of loss occurred when feeding supplements containing > or = 30% of DIP from urea 3 d/ wk. Cumulative BC change followed a similar trend. In conclusion, moderate protein (< or = 30% CP) supplements with < or = 30% of supplemental DIP from urea can be fed on alternate days without a substantive performance penalty. However, infrequent feeding of higher protein (> 30% CP) supplements with significant urea levels (> 15% of DIP from urea) may result in decreased performance compared with lower urea levels.

Ruminal and host adaptations to changes in frequency of protein supplementation.

The effect of altering supplementation frequency on host N balance and key N transactions in the ruminal ecosystem were monitored. Four ruminally fistulated beef steers (BW = 513 kg; SEM = 6.5) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Supplementation frequencies were 2 and 7 d/wk. Steers were fed tallgrass prairie hay (73.1% NDF, 5.3% CP) ad libitum. Supplement (42% CP; DM basis) was fed at 0.36% BW/d to steers supplemented 7 d/wk. Steers supplemented 2 d/wk received the same amount of supplement per week, but it was equally split among the two supplementation events. Steers supplemented 7 d/wk had higher forage (P < 0.02) and total digestible OM intake (P < 0.06), total N intake, fecal N excretion, and N retention. Although both supplementation frequencies were characterized by positive N balance, the decrease in N retention in the steers supplemented 2 d/wk was due to higher (P < 0.01) urinary N loss. Ruminal fluid was sampled at 0, 2, 4, 6, 12, 24, 48, and 72 h after supplementation beginning on a day when both treatments were supplemented. Frequency x hour interactions (P < 0.02) were observed for ruminal N metabolism criteria. Counts of peptide- and AA-fermenting bacteria peaked at 2 h and returned to nadir by 12 h for steers supplemented 7 d/wk. Steers supplemented 2 d/wk peaked at 6 h with a greater population and returned to nadir at 48 h. Ruminal ammonia concentrations followed a similar trend. Specific activity of ammonia production was lower (P < or = 0.05) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h was the same as for 7 d/wk steers. Ruminal peptides and free AA peaked at 2 h for steers supplemented 2 d/wk and were generally higher (P < or = 0.05) during the first 6 h compared with steers supplemented 7 d/wk. Total VFA concentration was not different (P = 0.35) due to supplementation frequency. Frequency x hour interactions (P < 0.01) were observed for all molar proportions of VFA. The molar proportion of acetate and acetate:propionate ratio were lower (P < 0.01) and the molar proportions of propionate and butyrate were higher for steers supplemented 2 d/wk from 4 h to 24 h. In conclusion, forage use and N balance improved with supplementation 7 d/wk, but supplementation 2 d/wk was associated with some desirable shifts in select ruminal events that may contribute to moderating potential negative impacts of supplementing infrequently.