Inclusion of shrub forages as a protein supplement in cattle feeding and its effect on live weight, body condition and milk production

Inclusão de forragens arbustivas como suplemento proteico na alimentação de bovinos e seu efeito sobre o peso vivo, a condição corporal e a produção de leite

Abstract

Livestock farming in Latin America, especially in the Amazon region, faces the challenge of moving towards more sustainable systems and reducing dependence on expensive concentrates. The objective of this research was to evaluate the inclusion of shrub forages as a protein supplement and its effect on live weight (LW), body condition score (BCS), and milk production in cattle. For this study, ten multiparous cows (Jersey, Holstein, and Jersey × Gyr crosses) at mid-lactation (± 180 days) were randomly assigned to two treatments with 15% inclusion of shrub: T1 (Gliricidia sepium) and T2 (Tithonia diversifolia). After this, under a 2 × 2 crossover design with a 7-day adaptation period followed by two experimental periods of 21 days each were applied. Data were analyzed using GLM and mixed models (SAS v.9.4). The results showed no significant variations (P > 0.05) in LW (445 ± 1 kg) or BCS (2.85 ± 0.45), suggesting adequate synchrony between available energy and microbial protein without mobilization of fat reserves. Average milk production was 6.54 ± 0.43 kg/day, with no statistical differences between treatments (P = 0.22), although a significant influence of the period was detected (P < 0.001). It is concluded that the use of local shrubs such as G. sepium and T. diversifolia represents a viable and economic nutritional alternative to maintain bovine productivity in smallholder systems.

Keywords:
dairy cattle; Gliricidia sepium; Tithonia diversifolia; shrub forages; milk production

Resumo

A pecuária na América Latina, especialmente na região amazônica, enfrenta o desafio de migrar para sistemas mais sustentáveis ​​e reduzir a dependência de concentrados caros. O objetivo desta pesquisa foi avaliar a inclusão de forragens arbustivas como suplemento proteico e seu efeito sobre o peso vivo (PV), o escore de condição corporal (ECC) e a produção de leite em bovinos. Para este estudo, dez vacas multíparas (Jersey, Holandesa e mestiças Jersey × Gir) em meados da lactação (± 180 dias) foram distribuídas aleatoriamente em dois tratamentos com 15% de inclusão de arbusto: T1 (Gliricidia sepium) e T2 (Tithonia diversifolia). Em seguida, foi aplicado um delineamento cruzado 2 × 2 com um período de adaptação de sete dias, seguido por dois períodos experimentais de 21 dias cada. Os dados foram analisados ​​utilizando GLM e modelos mistos (SAS v.9.4). Os resultados não mostraram variações significativas (P > 0,05) no peso vivo (445 ± 1 kg) ou na condição corporal (2,85 ± 0,45), sugerindo sincronia adequada entre a energia disponível e a proteína microbiana, sem mobilização das reservas de gordura. A produção média de leite foi de 6,54 ± 0,43 kg/dia, sem diferenças estatísticas entre os tratamentos (P = 0,22), embora tenha sido detectada influência significativa do período (P < 0,001). Conclui-se que o uso de arbustos locais, como G. sepium e T. diversifolia, representa uma alternativa nutricional viável e econômica para manter a produtividade bovina em sistemas de pequenos produtores.

Palavras-chave:
bovinos leiteiros; Gliricidia sepium; Tithonia diversifolia; forragens arbustivas; produção de leite

1. Introduction

According to demographic projections from the Food and Agriculture Organization of the United Nations FAO (2022), the world population is estimated to reach approximately 9.3 billion people by 2050. This population growth places unprecedented pressure on agri-food systems, which will need to increase their production by more than 60% to meet the growing demand for food (Michalk et al., 2019). In this context, the agricultural sector faces the challenge of improving its productive efficiency while simultaneously ensuring environmental sustainability and global food security.

In the context of both dairy and beef cattle farming, focusing on the differences between livestock systems, these represent a vital link in social and economic terms, as they contribute directly to the livelihoods of millions of families (FAO, 2019; Montenegro and Abarca, 2021; Moorby and Fraser, 2021), and play a key role in providing ecosystem services such as soil fertility, landscape conservation, and the regulation of biogeochemical cycles (Montenegro and Abarca, 2021; Moorby and Fraser, 2021). However, in the face of climate change and despite its economic and environmental importance, the livestock sector is facing increasingly complex challenges.

To tackle these challenges, the United Nations (UN) has set out 17 Sustainable Development Goals (SDGs) as a strategy for the public and private sectors to adopt in order to tackle and eradicate hunger, reduce inequalities and mitigate the effects of climate change (ONU, 2020). Although many argue that livestock farming is responsible for a large proportion of greenhouse gas emissions (Durmic et al., 2025), of which methane is by far the most significant, there is no doubt that livestock farming systems make a significant contribution to food security and rural development (Cheng et al., 2022; Martínez-Salinas et al., 2024). In this regard, estimates indicate that the livestock sector accounts for 12 to 14% of greenhouse gas emissions, with enteric fermentation in ruminants being one of the main sources. However, of this total, between 3% and 4% is attributed to dairy farming, while 12% to 13% corresponds to meat production (Moorby and Fraser, 2021). Given this context, implementing strategies to reduce the livestock sector’s carbon footprint is a key element and a constant topic of research.

In this regard, a growing number of studies have focused on developing strategies to modulate the rumen ecosystem, aiming to improve digestive efficiency and reduce enteric methane production. Among these strategies, the use of diets with higher nutritional quality and digestibility stands out, as it not only optimizes nutrient utilization but also contributes to reducing greenhouse gas emissions (Silvestre et al., 2021; Stanley et al., 2018). However, implementing these strategies presents particular challenges in regions like Latin America, where extensive livestock systems predominate, characterized by the use of natural pastures or monocultures and limited dietary diversification (Rocha et al., 2019).

Therefore, as affirmed by Thornton and Gerber (2010) , it is essential to promote the transition to more sustainable, resilient production systems adapted to local conditions. This transition involves not only the adoption of innovative technologies but also the revaluation of alternative forage resources that improve production efficiency and reduce environmental impact (Biglari et al., 2019; FAO, 2018). So, the use of alternative feed sources for ruminants has sparked growing interest in the scientific community (Leng et al., 2011; Manuelian et al., 2021), especially those based on native plant species or those adapted to specific agroecological conditions.

In tropical livestock systems, where conventional protein supplements such as soybean meal and corn are often economically inaccessible for smallholder farmers, shrub forages have emerged as a strategic nutritional alternative. Species such as Gliricidia sepium and Tithonia diversifolia are particularly well-suited to tropical agroecologies due to their rapid biomass production, drought tolerance, and ability to thrive on marginal soils. G. sepium, a multipurpose leguminous tree, is recognized for its high crude protein content (18-28% DM), favorable amino acid profile, and presence of condensed tannins that can improve protein utilization by reducing ruminal degradation without compromising fiber digestibility when fed at moderate inclusion levels (<30%) (Alamu et al., 2023; Aregheore and Perera, 2004; Shem et al., 2003). Similarly, T. diversifolia is a fast-growing, high-yielding shrub with crude protein levels ranging from 15 to 24% DM, rich in essential minerals and secondary metabolites with potential antiparasitic and antioxidant properties. Field and experimental studies across Latin America, Africa, and Southeast Asia have demonstrated that incorporating these shrubs into ruminant diets can maintain or improve dry matter intake, support ruminal microbial efficiency, and sustain milk yield while significantly reducing feed costs (Hahn-Von-Hessberg et al., 2016; Ribeiro et al., 2015; Rivera et al., 2018). However, their application in the Ecuadorian Amazon, a region characterized by high rainfall, humid tropical conditions, and fragmented smallholder dairy systems, remains underexplored. Furthermore, optimal inclusion levels and their direct effects on productive and physiological parameters under local management practices require empirical validation. Therefore, this study evaluates the inclusion of 15% G. sepium and T. diversifolia shrub as protein supplements in mid-lactation dairy cows, assessing their impact on live weight, body condition score, and milk production in a smallholder tropical context.

2. Materials and Methods

2.1. Ethical approval and study site

All experimental procedures were conducted in accordance with national guidelines for animal welfare and research (AGROCALIDAD, 2022) and were approved by the Institutional Animal Care and Use Committee. The study was carried out in Unión Milagreña parish, Orellana province, Ecuador (0°21’47”S, 76°47’04”W; 265 m a.s.l.), characterized by a tropical humid climate. Ambient temperature and relative humidity were recorded daily using a calibrated digital datalogger placed at animal height within the management area (Figure 1).

Figure 1
Geographic location of the experiment.

2.2. Animals and treatments

Ten multiparous dairy cows in mid-lactation (180 ± 6 days in milk) were used, comprising three Jersey (490 ± 6 kg initial LW; 6.0 ± 0.3 kg/d milk), four Holstein (530 ± 8 kg; 7.5 ± 0.4 kg/d milk), and three Jersey × Gyr crosses (510 ± 5 kg; 8.0 ± 0.5 kg/d milk). Cows were allocated to a 2 × 2 crossover design (treatment × period; Figure 2) with two groups of five animals each. Group 1 received Treatment 1 (T1) during Period 1 and Treatment 2 (T2) during Period 2, while Group 2 received the reverse sequence. Each experimental period lasted 21 days, consisting of 14 days for dietary adaptation and 7 days for data collection. Prior to the trial, all animals underwent a 7-day baseline adaptation period on a Panicum maximum + Arachis pintoi pasture (80:20) with ad libitum water access.

Figure 2
Experimental design used to evaluate two management strategies.

2.3. Dietary treatments and feeding management

Two experimental diets were formulated using software to meet the nutrient requirements of mid-lactation dairy cows (~12% CP, 1.55 Mcal ME/kg DM) (NASEM, 2021). Both diets contained 15% shrub meal (dry matter basis) as the protein supplement: T1 included Gliricidia sepium, and T2 included Tithonia diversifolia. The basal composition was: King grass (65%), sugarcane (Saccharum officinarum, 5%), cracked corn (Zea mays, 15%), bypass fat (0.3%), mineral-vitamin premix (0.1%), and sodium bicarbonate (0.05%). Shrub foliage was harvested fresh, shade-dried for 72 h, ground to 2 mm, and uniformly mixed into the total ration. Diets were offered twice daily at (07:00h). Proximate chemical composition of all ingredients and final diets was determined following AOAC (2000). Crude protein, 15.83%; Neutral detergent fiber, 35%; Acid detergent fiber, 35%, No-fiber carbohydrates 40%; ethereal extract, 2%; minerals, 1%.

2.4. Experimental measurements

2.4.1. Live weight and body condition score

Live weight (LW) was measured for all animals at the beginning and end of each 21-day experimental period using a bovine weight tape (900 kg capacity, ±2 kg accuracy) wrapped around the thoracic perimeter. Body condition score (BCS) was assessed for all cows at the same time points following the methodology of Russel et al. (1969) and Morgan-Davies et al. (2008), using a 1-5 scale where 1 represents an emaciated animal and 5 represents an obese animal. All assessments were performed by the same trained evaluator to minimize subjective variation.

2.4.2. Milk production

Cows were milked once daily at 08:00h using a portable milking unit (Westfalia-Separator Ibérica, Granollers, Spain) operated at 42 kPa vacuum pressure, 90 pulsations/min, and a 66% pulsation ratio. The milking routine included attaching the cluster, machine milking, machine unmilking before removing the cluster, and dipping the teats in an iodine solution (P3-ioshield, Ecolab Hispano-Portuguesa; Barcelona, Spain). Daily milk yield was recorded at each milking using calibrated volumetric jugs (22 L capacity, ±1% accuracy), with values logged to the nearest 0.1 kg. To account for daily variation, production data were collected throughout the 7-day measurement phase of each experimental period (days 15-21), and individual cow means were calculated as the average of the seven daily records.

Daily milk yield was recorded at each milking using calibrated volumetric jugs (22 L capacity, ±1% accuracy), with values logged to the nearest 0.1 kg. To account for daily variation, production data were collected throughout the 7-day measurement phase of each experimental period (days 15-21), and individual cow means were calculated as the average of the seven daily records.

2.3. Statistical analysis

All data were previously subjected to a Shapiro-Wilk normality test (live weight [LW] and body condition score [BCS]), while milk production was analyzed using the Kolmogorov-Smirnov test. Once data normality was confirmed (P > 0.05), LW and BCS were processed using a general linear model (GLM), while milk production was processed using a mixed linear model for repeated measures (MIXED), in the statistical software SAS v. 9.4 (SAS Inst. Inc.; Cary, North Carolina, USA). Means are presented as least squares and were compared using a Tukey t-test. Statistical differences were considered when the P-value was < 0.05.

3. Results

The live weight of the animals according to the administered protein strategy and period is shown in Figure 3. No variation in live weight was detected with respect to the feeding strategy (445 ± 1 kg; P = 0.99). Similarly, despite the slight numerical differences observed between periods regarding live weight, these were not significant (P = 0.32), corresponding to the lack of interaction observed between treatment and period (P = 0.76).

Figure 3
Evolution of live weight (LW, kg) of mid-lactation dairy cows fed diets supplemented with 15% Gliricidia sepium and Tithonia diversifolia in a 2 × 2 crossover design (n = 10 cows). Data represent least squares means ± SEM.

Similar results were observed for body condition score (BCS; Figure 4). As a subjective method for assessing body reserves, BCS is widely used in dairy production. In this study, given the nature of the experimental design used (crossover), BCS showed no variation due to the period effect (2.87 ± 0.45; P = 0.99), treatment effect (2.85 ± 0.45; P = 0.84), or the treatment × period interaction (P = 0.38).

Figure 4
Body condition of animals as a function of fed diets supplemented with 15% Gliricidia sepium and Tithonia diversifolia.

Milk production data according to the main effects considered in this study are shown in Table 1. No significant differences (P = 0.22) were observed between treatments in the average daily milk production (6.54 ± 0.43 kg/day). However, the study period influenced the amount of milk produced; in fact, 12% more milk was obtained in the first period than in the second (6.96 vs. 6.12 ± 0.43 kg/day; P < 0.001). Although each 21-day period included a 14-day adaptation phase that may have partially mitigated residual effects, we cannot fully exclude the possibility of carryover influencing the observed period effect on milk production. Future studies evaluating shrub forages in tropical dairy systems should incorporate a formal washout period (e.g., 7-10 days on basal diet) to isolate treatment effects from temporal or carryover influences. Nevertheless, the non-significant treatment × period interaction (P = 0.35) and the inclusion of sequence as a fixed effect in the statistical model provide some confidence that the primary conclusion equivalence between G. sepium and T. diversifolia at 15% inclusion remains robust. No differences were detected for the treatment × period interaction (P = 0.35). In contrast, the treatment × week interaction showed a significant variation in average daily production.

Table 1
Least squared means of milk production according to the main effects.

Item	Period	SEM	P = value
	Gliricidia	Tithonia	1	2		Treat	Period	P × T	T × W
Milk production, kg/d	6.60	6.48	6.96a	6.12b	0.43	0.22	0.001	0.35	0.001
Fat, %	2.72	2.72	1.97	3.52	0.18	0.69	0.001	0.89	0.007
Protein, %	3.42	3.33	3.39	3.36	0.05	0.15	0.59	0.39	0.24
FCM 4%1, kg/d	5.02	5.16	4.61	5.57	0.29	0.38	0.001	0.29	0.001

• ¹
  Milk corrected to 4% = milk × (0.15 × % fat + 0.4); SEM, standard error of the mean; P, period; T, Treat; W, week.
• ^a-bMeans with different letters in the same row indicate significant differences at P < 0.05.

When we analyzed the 4% standardized milk across treatments, the results showed no significant differences (P = 0.38). However, highly significant differences were observed (4.61 vs. 5.57 ± 0.29 kg/day; P < 0.001; Figure 5) for the period effect. Despite this, the period × treatment interaction was not significant (P = 0.29), although there was a strong interaction between treatment × week (P < 0.001). It is important to note that, although a moderate correlation with ambient temperature was observed (r = 0.58-0.63), the result was not significant (P = 0.39-0.42).

Figure 5
Fat-corrected milk for the treatment period.

4. Discussion

Russel et al. (1969) who initially devised this methodology for sheep production, now widely used in the livestock industry, consider it a subjective method that is very useful because it allows us to assess body reserves and thus apply corrective measures in nutritional management. In this study, body condition score (BCS) was constant and unassociated with the animals’ live weight (r = 0.10; P = 0.40), indicating these fluctuations are negligible and did not mobilize fat tissue to meet the animals’ energy needs (Berry et al., 2007).

According to official statistics from the Ecuadorian Ministry of Agriculture and Livestock (Ecuador, 2022), the national average milk production in dual-purpose and smallholder dairy systems is approximately 5.0 L/day/cow, which includes both specialized dairy breeds and crossbred animals managed under extensive to semi-intensive conditions. In our study, although the environmental conditions were not optimal for Bos taurus cattle, the average daily milk production we observed showed a slight numerical increase compared to the national data. Furthermore, extensive scientific evidence has already revealed a strong influence of temperature and relative humidity on milk production (Mehaba et al., 2021), however, this study showed weak (r = 0.10-0.30) but statistically significant (P = 0.05-0.10) correlations.

Studies conducted by Arias et al. (2018), replacing 50% of the feed with Tithonia spp. instead of balanced feed, observed a decrease in milk production when compared to a control group of 100% balanced feed and 25% Tithonia. These results agree with our work, which found that since the supplementation with this shrubby forage was no greater than 25%, no significant differences were observed. Ribeiro et al. (2016) supplemented Holstein × Zebu cows with 15% Tithonia spp. and had no effect on milk production. Consequently, this species, as an alternative forage, could provide crude protein for ruminant feed, in addition to offering environmental benefits due to its ability to absorb nitrogen in the soil and improve its physical qualities (Mauricio, 2017). Therefore, this study indicates that fresh T. diversifolia at up to 15.3% in the diet of dairy cows can replace sugarcane but also conventional feed supplements (soybean and corn) without any change in intake, milk production/composition, and most blood parameters.

An interesting study by Clavero et al. (1996) in which dairy cows were supplemented with 33% Glyricide spp. meal, showed a numerical decrease in milk production compared to levels of 16%. Similarly, a study conducted on Jersey cows at peak lactation (2^nd and 3^rd months) compared several shrub legumes, highlighting Glyricide spp. in terms of milk production. Along the same lines, another study in Jersey cows by Sharma and Ingalls (2022) administered Glyricide spp. at levels ranging from 0 to 30% and observed no differences in feed intake parameters or average daily milk production.

The absence of negative effects on milk production at 15% inclusion of G. sepium and T. diversifolia can be explained by several complementary physiological mechanisms. First, both shrub species exhibit moderate levels of condensed tannins (3-6% DM), which at this concentration enhance metabolizable protein supply by reducing ruminal protein degradation without compromising fiber digestibility or voluntary intake (Frutos et al., 2004). This “protein bypass” effect likely improved the post-ruminal amino acid flow, compensating for any potential reduction in ruminal ammonia availability. Second, the degradability kinetics of these shrubs characterized by relatively low NDF (38-39% DM) and ADF (18-23% DM) compared to tropical grasses suggest rapid initial fermentation that provides readily available energy for microbial growth during the first hours post-feeding (Morais et al., 2023). This temporal synchrony between energy release (from rapidly fermentable carbohydrates in shrub leaves) and nitrogen availability (from both rumen-degradable and bypass protein fractions) likely sustained microbial protein synthesis efficiency (Esposito et al., 2014). Third, the maintenance of body condition score and live weight throughout the trial indicates that energy balance remained positive or at equilibrium, preventing the mobilization of body reserves that would otherwise signal nutritional inadequacy (D’Occhio et al., 2019). The inclusion of bypass fat (0.3%) and cracked corn (15%) in the basal diet provided sufficient glucogenic precursors to support lactose synthesis, while the shrub forages contributed high-quality protein without excessive ruminal ammonia production or energy expenditure for urea excretion (Rodríguez-Prado et al., 2010). Finally, secondary metabolites present in Gliricidia and Tithonia (including flavonoids and saponins) may have exerted subtle modulatory effects on rumen fermentation, potentially improving propionate:acetate ratios or reducing methanogenesis, although these parameters were not measured in the present study. Future research should include in vivo rumen fermentation profiling (VFA proportions, pH, ammonia-N) and blood metabolite analysis (urea, glucose, β-hydroxybutyrate) to confirm these hypothesized mechanisms.

Consequently, all these responses obtained regarding milk production could be explained by their low NDF (38-39% on a dry matter basis) and ADF (13-23% on a dry matter basis) contents, which have resulted in dry matter degradation percentages ranging from 71-81%, as reported by Morais et al. (2023). Therefore, the rising price of concentrates limits their supplementation in cattle fed on low-quality pastures. In the case of the Orellana province, the high cost of concentrated feeds for small farmers hinders the optimization of livestock productivity. For this reason, based on this research, the use of local shrub forages would be a viable alternative for ruminant feeding, providing cheaper and more nutritious feed resources.

5. Conclusions

The evolution of live weight and body condition score (BCS) in cows subjected to different protein supplementation strategies revealed a possible synchrony between the amount of microbial protein and available energy, which helped prevent changes in the animals’ body reserves. Slight numerical differences in milk production were observed when comparing Gliricia sepium vs. Tithonia diversifolia, but we highlight a strong influence of the supplementation period, which could affect the persistence of lactation in dairy cows under this protein supplementation.

Data Availability Statement

Research data is only available upon request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  24 July 2026
• Date of issue
  2026

History

• Received
  31 Mar 2026
• Accepted
  17 May 2026