Dynamics of rumen microbial protein synthesis and purine derivative excretion in HF-crossbred and Deoni cows subjected to Prepartum energy fortification

Introduction

Rumen microbial protein (MBP) typically supplies 50-80% of the metabolisable protein reaching the small intestine of the dairy cow, making the efficiency of its synthesis a firstorder determinant of protein economy, particularly during the transition period when protein demand for colostrogenesis, fetal growth and the onset of lactation rises sharply against a background of falling or slowly recovering dry matter intake (Bach et al., 2005; Hackmann and Firkins, 2015) [1,11] . Because direct measurement of duodenal microbial flow requires cannulated animals, urinary excretion of purine derivatives (PD)allantoin and uric acid, the end products of microbial nucleic acid degraded and absorbed in the small intestinehas become the standard non-invasive proxy for MBP supply in both research and applied settings (Chen and Gomes, 1992; Chen et al., 1990) [3,4] . Recent reviews continue to highlight that, notwithstanding its wide adoption, the PD technique carries substantial experimental error and that newer molecular approaches to characterising the rumen microbiome may eventually improve on it, though the urinary PD method remains the most practical field-applicable tool available for breed-and treatment-comparative work of the kind reported here (Lima et al., 2023) [14] .

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com Breed differences in MBP synthesis are well documented in the comparative Bos indicus-Bos taurus literature. Indicine and crossbred cattle have repeatedly been shown to differ from taurine breeds in urinary purine derivative excretion per unit of intake, in nitrogen and urea recycling, and in the endogenous purine contribution to excreted purine derivatives, reflecting differences in rumen volume, intake capacity, digesta kinetics and purine metabolism between genotypes (Zhou et al., 2017; Prates et al., 2012; Gandra et al., 2011) [20,17,9] . A recent systematic review of the purinederivative literature likewise reported systematically different purine-derivative excretion per unit of dry matter intake between Bos indicus and Bos taurus groups (Lima et al., 2023) [14] . Because rumen size scales with body size and MBP output scales, in turn, with the volume of fermentable substrate processed, larger-framed crossbred cattle would be expected to produce more MBP in absolute terms than smaller indigenous cattle on a similar diet; genotype differences in digestive-tract function of this kind have been reviewed in detail elsewhere (Hegarty, 2004) [12] . Whether the larger absolute MBP output of larger-framed cattle translates into a proportionate advantage in the efficiency of MBP synthesis per unit of substrate fermentedthe metric of greatest nutritional and economic relevanceis a distinct question that has received little attention in a periparturient, breed-comparative framework. This paper addresses that question using a controlled prepartum energy-fortification trial in HF-crossbred (HFX) and Deoni cows in which urinary PD excretion, PD index, duodenal MBP flow and MBP production efficiency (g/kg digestible organic matter intake, DOMI, and g/Mcal metabolisable energy, ME) were measured in both the prepartum close-up period and again postpartum. The specific objectives were: (i) to quantify breed differences in PD excretion and MBP synthesis in each period; (ii) to characterise how prepartum energy source (control, glycerol, jaggery, maize) and functional nutrient supplementation (FNS) modulated these indices within each breed; and (iii) to compare the prepartum-to-postpartum trajectory of MBP synthesis and its efficiency between breeds, testing whether the larger absolute MBP output of HFX cows is matched by a proportionate efficiency advantage across the transition.

Materials and Methods

Animals, location and design

Purine derivative sampling and analysis

Spot urine samples were collected by free-catch or gentle vulval stimulation at the close-up prepartum timepoint and again during the postpartum period, and analysed for allantoin, uric acid and creatinine by standard colorimetric/HPLC-based methods following the principles of Chen and Gomes (1992) [3] . Total PD was calculated as the sum of allantoin and uric acid; PD index was derived by relating PD concentration to estimated urine volume using the creatinine: body-weight relationship, an approach whose accuracy relative to total urine collection has been examined directly in grazing cattle (Moorby et al., 2006; Da Silva Júnior et al., 2021) [16,6] ; and daily PD excretion (mmol/d) and duodenal MBP flow (g/d) were computed from PD excretion using standard purine-derivative equations for cattle (Chen and Gomes, 1992; Belenguer et al., 2002; González-Ronquillo et al., 2003) [3,2,10] , following the technical approach applied more recently to beef and dairy cattle by Crawford et al. (2020) [5] and validated for dairy diets of differing forage proportion by Moorby et al. (2006) [16] and for diets of differing quality by Martínez et al.

(2010) [15] . MBP production efficiency was expressed as g MBP per kg digestible organic matter intake (DOMI) and as g MBP per Mcal metabolisable energy (ME) intake, using digestible OM and ME intake values derived from the parallel intake and digestibility measurements taken on the same animals.

Statistical analysis

Data were analysed by 2 × 2 × 4 factorial ANOVA (breed, FNS, energy source as fixed effects) separately for the prepartum and postpartum periods, with prepartum-topostpartum change computed as the percentage difference between period means for each breed × treatment cell.

Least-squares means±SEM are reported; treatment means were separated by Duncan's Multiple Range Test; significance was declared at P≤0.05 and tendencies at P ≤ 0.10.

Results and Discussion

Breed differences in purine derivative excretion

Urinary allantoin excretion was significantly higher in HFX than Deoni cows in both the prepartum (4.97 vs. 4.37 mmol/L; p < 0.01) and postpartum (4.79 vs. 4.17 mmol/L; p < 0.001) periods (Table 1). Allantoin comprised a larger share of total PD in HFX cows (91% prepartum, 86% postpartum) than in Deoni cows (85% prepartum, unchanged postpartum), with the balance made up by uric acida pattern consistent with the 80-85%:15-20% allantoin:uric-acid partition reported for cattle generally (Chen et al., 1990) [4] . Total PD concentration was higher in HFX than Deoni cows in both periods, reaching significance postpartum (p < 0.001) and showing a tendency prepartum (P = 0.066). PD index and daily PD excretion were both significantly higher in HFX than Deoni cows in each period (p < 0.001), and neither showed significant interaction with energy source, FNS supplementation, or their combination -PD excretion was, in this dataset, essentially a pure breed effect, consistent with the parent thesis finding that PD excretion rate differs by breed independent of dietary energy manipulation. This is consistent with breed differences in PD excretion being driven primarily by structural factorsrumen volume, absolute digestible substrate throughput and glomerular filtration raterather than by the composition of prepartum energy fortification, a conclusion supported both by comparative digestion studies across Holstein, Nellore and buffalo genotypes (Gandra et al., 2011) [9] and by a recent review of genotype effects on digestive-tract function in ruminants (Hegarty, 2004) [12] .

Duodenal MBP flow: A rumen-size-driven breed effect

Daily duodenal MBP flow was 35% higher in HFX than Deoni cows during the prepartum period (350.1 vs. 259.1 g/d; p < 0.001) and remained significantly higher (43% relatively) postpartum (327.1 vs. 229.1 g/d; p < 0.001) (Fig. 1; Table 1). Because digestible DM or OM intake in absolute terms was consistently higher in HFX cowsdespite Deoni cows showing higher intake per unit metabolic body weight, as reported in the companion comparative-intake analysis of this cohortand because MBP synthesis scales with the absolute quantity of fermentable substrate processed rather than with substrate intake normalised to body size, the larger metabolic body size of HFX cows (26% greater than Deoni in this cohort) was the principal driver of their higher absolute MBP output. This scaling of duodenal microbial nitrogen flow with the volume of substrate processed has been demonstrated directly in calves using duodenal cannulation alongside urinary PD estimates (Funaba et al., 1997) [8] , and is consistent with the general principle that MBP production scales with rumen size and fermentable substrate throughput.

Energy source and functional supplement effects within breed

Across the four prepartum energy sources, PD excretion and duodenal MBP flow showed the expected breed separation within every energy group (Table 2; Fig. 3 showed more overlap between breeds by energy source, with Deoni cows under jaggery (77.8 g/kg DOMI) essentially matching or exceeding HFX cows under glycerol (77.5 g/kg DOMI)an early signal, even within the prepartum period, that Deoni MBP synthesis was not simply a scaled-down version of the HFX pattern but showed some energy-source-specific efficiency advantages of its own. Formal ANOVA confirmed that energy source alone did not significantly affect PD excretion, PD index or duodenal MBP flow, nor did the energy × breed, energy × FNS, or three-way interactions reach significance for these traitsconsistent with the parent dataset's finding that PD excretion was essentially a pure breed effect. MBP efficiency (g/kg DOMI), by contrast, did show a significant effect of energy source (P = 0.02 prepartum), with jaggery and maize numerically outperforming glycerol, plausibly reflecting the more rapid ruminal availability of jaggery's simple sugars and maize starch relative to the additional phosphorylation step required to route glycerol into central fermentative metabolism, a rate-limiting step also implicated in reduced ruminal digestibility responses to glycerol supplementation in cattle (Wang et al., 2009) [19] . Prepartum FNS supplementation increased postpartum MBP production by approximately 7% in both breeds, though this improvement in absolute output was not matched by a statistically significant improvement in MBP efficiency, indicating that the FNS benefit operated mainly through increased substrate throughput rather than through improved conversion efficiency per unit of substrate fermented.

The prepartum-to-postpartum efficiency reversal

The most striking finding of this analysis is a reversal in relative MBP efficiency ranking between breeds across the transition (Fig. 2; Table 3 This asymmetric decline is consistent with the substantially larger postpartum surge in intake documented in HFX cows in this cohorta 74% rise in total diet DMI per unit metabolic body weight from prepartum to postpartum, against only a 12% rise in Deoni cows (reported in the companion comparative-intake analysis of this cohort)which would be expected to push a larger, more rapidly changing volume of fermentable substrate through the rumen without a proportionate increase in microbial growth, consistent with the general principle that rapid increases in fermentable carbohydrate availability can depress microbial growth efficiency through energy-spilling and futile-cycle mechanisms in mixed rumen microbial populations (Hackmann and Firkins, 2015) [11] . This general pattern of shifting digestive and metabolic efficiency across the dry-tolactation transition, including changing energy and nitrogen balance from the dry period into early lactation, has also been documented directly using total-collection balance trials in dairy cows (Daniel et al., 2022) [7] . All postpartum MBP efficiency values in both breeds nonetheless remained well above the approximately 30 g/kg DOM optimum reported for continuous-culture rumen fermentation systems (Hackmann and Firkins, 2015) [11] , indicating that neither breed's rumen microbial population was functioning inefficiently in absolute termsthe reversal is a relative, between-breed phenomenon rather than evidence of dysfunction in either group. Both allantoin and total PD showed relatively small prepartum-to-postpartum declines (Fig. 4; Table 3) in contrast to the much larger declines in PD index, PD excretion, duodenal MBP flow and MBP efficiency, a pattern attributable in part to elevated prepartum plasma and urinary allantoin arising from fetal and whole-body nucleic acid turnover in the last weeks of gestation, a phenomenon reported to inflate apparent MBP estimates during the final eight weeks of pregnancy before falling sharply after calving. Creatinine and uric acid excretion rose more in HFX (+ 26% and + 65%, respectively) than in Deoni cows (+ 8% each) from prepartum to postpartum, consistent with the larger increase in muscle metabolic turnover and absolute digestible substrate flow accompanying the substantially larger postpartum intake surge in HFX cows documented above.

Integrative interpretation and practical implications

Taken together, these results indicate that breed shapes microbial protein synthesis in two distinct ways that point in different directions. In absolute terms, the larger-framed HFX genotype consistently out-produced the smaller Deoni genotype in PD excretion and duodenal MBP flow in both the prepartum and postpartum periods, a rumen-size and substrate-throughput effect that is unsurprising and consistent with the wider comparative-nutrition literature. In relative, efficiency terms, however, the postpartum period exposed an asymmetry: HFX cows paid a much larger efficiency penalty (43% decline in g/kg DOMI) than Deoni cows (18% decline) for their much larger postpartum intake surge, to the point of falling behind Deoni cows in DOMIreferenced MBP efficiency after calving despite having started the prepartum period with a significant efficiency advantage.

From a practical protein-nutrition standpoint, this suggests that the rapid postpartum ration changes typically applied to high-yielding crossbred cows to meet their much larger absolute nutrient demand may be outpacing the rumen microbial population's capacity to convert that additional substrate into protein with matching efficiency, at least during the acute early-lactation adjustment window captured here. Where feasible, a more graduated postpartum transition in fermentable carbohydrate and protein supply for HFX-type cowsrather than the sharp step-up documented in this cohortcould help narrow the efficiency penalty and reduce the volume of degraded protein and carbohydrate lost to inefficient microbial metabolism rather than converted to usable microbial protein. For indigenous Deoni cows, by contrast, the comparatively stable MBP efficiency across the transition suggests less urgency for postpartum ration graduation on this specific axis, consistent with the more modest and better-matched postpartum intake response documented in this breed.

Limitations should be noted. PD-based MBP estimates in the prepartum period are subject to a well-documented upward bias from fetal and whole-body nucleic acid turnover in late gestation, meaning that absolute prepartum MBP efficiency values (up to 80 g/kg DOMI, well above even the top of the theoretical in vivo range of 34-66% of maximum microbial growth efficiency reported elsewhere (Hackmann and Firkins, 2015)) [11] should be interpreted as relative rather than absolute estimates, as is standard practice in the PD-technique literature (Chen and Gomes, 1992) [3] . Because spot urine sampling and the creatinine: body-weight assumption were used to estimate daily urine volume rather than total urine collection, some estimation error is expected; work directly comparing spot-sample and total-collection PD estimates, and comparing collection days and times, indicates that such error, while non-trivial, would be expected to affect both breeds similarly and should not materially alter the relative breed comparison that is the focus of this paper (Da Silva Júnior et al., 2021; Silva Júnior et al., 2018) [6,18] . A dedicated study incorporating total urine collection and duodenal cannulation across the transition period would be needed to confirm the magnitude of the efficiency reversal identified here with greater precision.

Conclusion

HF-crossbred cows produced significantly more microbial protein than Deoni cows in absolute terms throughout the transition period, consistent with their larger rumen size and greater absolute digestible substrate intake. However, this absolute advantage was not matched by a proportionate efficiency advantage after calving: MBP production efficiency per unit of digestible organic matter intake declined by 43% in HFX cows postpartum against only 18% in Deoni cows, reversing the prepartum efficiency ranking between breeds. Energy source had limited effect on PD excretion or duodenal MBP flow within breed, but did significantly affect MBP efficiency, and prepartum functional nutrient supplementation modestly increased postpartum MBP output in both breeds. These findings identify the postpartum period as a window of disproportionate microbial-protein-synthesis efficiency cost in high-yielding crossbred cows relative to indigenous cattle, and support more graduated postpartum ration transitions as a potential strategy to narrow this efficiency gap in tropical mixed-breed dairy systems.

Acknowledgement

AcknowledgmentsThe authors thank the Director, ICAR-National Dairy Research Institute, and the Officer-in-Charge, ICAR-NDRI Southern Regional Station, Bengaluru, for the institutional facilities provided to conduct this study, and the farm and laboratory staff of the Livestock Research Centre for their assistance with animal management and sample analysis.

References

1. Bach A, Calsamiglia S, Stern MD. Nitrogen metabolism in the rumen. J Dairy Sci. 2005;88(E Suppl):E9-E21.
2. Belenguer A, Yañez D, Balcells J, Ozdemir NH, González-Ronquillo M. Urinary excretion of purine derivatives and prediction of rumen microbial outflow in goats. Livest Prod Sci. 2002;77(2-3):127-135.
3. Chen XB, Gomes MJ. Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives-an overview of the technical details. Occasional Publication. Aberdeen: International Feed Resources Unit, Rowett Research Institute; 1992.
4. Chen XB, Hovell FDD, Ørskov ER, Brown DS. Excretion of purine derivatives by ruminants: effect of exogenous nucleic acid supply on purine derivative excretion by sheep. Br J Nutr. 1990;63(1):131-142.
5. Crawford GI, MacDonald JC, Watson AK, Erickson GE, Klopfenstein TJ. Diurnal and dietary impacts on estimating microbial protein flow from urinary purine derivative excretion in beef cattle. Transl Anim Sci. 2020;4(3):txaa140.
6. Da Silva Júnior JM, Rodrigues JPP, Valadares Filho SC, Detmann E, Paulino MF, Rennó LN. Estimating purine derivatives and nitrogen compound excretion using total urine collection or spot urine samples in grazing heifers. J Anim Physiol Anim Nutr. 2021;105(5):861-873.
7. Daniel JB, Sanz-Fernandez MV, Nichols K, Doelman J, Martín-Tereso J. Digestive and metabolic efficiency of energy and nitrogen during lactation and the dry period in dairy cows. J Dairy Sci. 2022;105(12):9564-9580.
8. Funaba M, Kagiyama K, Iriki T, Abe M. Duodenal flow of microbial nitrogen estimated from urinary excretion of purine derivatives in calves after early weaning. J Anim Sci. 1997;75(7):1965-1973.
9. Gandra JR, Freitas JE, Barletta RV, Filho MM, Gimenes LU, Vilela FG, et al. Productive performance, nutrient digestion and metabolism of Holstein (Bos taurus) and Nellore (Bos taurus indicus) cattle and Mediterranean buffaloes (Bubalus bubalis) fed with corn-silage based diets. Livest Sci. 2011;140(1-3):283291.
10. González-Ronquillo M, Balcells J, Guada JA, Vicente F. Purine derivative excretion in dairy cows: endogenous excretion and the effect of exogenous nucleic acid supply. J Dairy Sci. 2003;86(4):1282-1291.
11. Hackmann TJ, Firkins JL. Maximizing efficiency of rumen microbial protein production. Front Microbiol. 2015;6:465.
12. Hegarty RS. Genotype differences and their impact on digestive tract function of ruminants: a review. Aust J Exp Agric. 2004;44(5):459-467.
13. ICAR. Nutrient Requirements of Cattle and Buffalo. 2nd ed. New Delhi: Indian Council of Agricultural Research; 2013.
14. Lima J, Ingabire W, Roehe R, Dewhurst RJ. Estimating microbial protein synthesis in the rumen-can 'omics' methods provide new insights into a long-standing question? Vet Sci. 2023;10(12):679.
15. Martínez ME, Ranilla MJ, Tejido ML, Ramos S, Carro MD. Urinary excretion of purine derivatives, microbial protein synthesis, nitrogen use, and ruminal fermentation in sheep and goats fed diets of different quality. J Dairy Sci. 2010;93(8):3684-3698.
16. Moorby JM, Dewhurst RJ, Evans RT, Danelón JL. Effects of dairy cow diet forage proportion on duodenal nutrient supply and urinary purine derivative excretion. J Dairy Sci. 2006;89(9):3552-3562.
17. Prates LL, Valadares Filho SC, Detmann E, Valadares RFD, Santos SA, Braga JMS, et al. Endogenous fraction and urinary recovery of purine derivatives in Nellore and Holstein heifers with abomasal purine infusion. Livest Sci. 2012;150(1-3):179-186.
18. Silva Júnior JM, Rennó LN, Valadares Filho SC, Paulino MF, Detmann E, Menezes GCC, et al. Evaluation of collection days and times to estimate urinary excretion of purine derivatives and nitrogen compounds in grazing Nellore cattle. Livest Sci. 2018;217:85-91.
19. Wang C, Liu Q, Huo WJ, Yang WZ, Dong KH, Huang YX. Effects of glycerol on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers. Livest Sci. 2009;121(1):15-20.
20. Zhou JW, Zhong CL, Liu H, Degen AA, Titgemeyer EC, Ding LM, et al. Comparison of nitrogen utilization and urea kinetics between yaks (Bos grunniens) and indigenous cattle (Bos taurus). J Anim Sci. 2017;95(10):4600-4612.