Effect of Supplementation on the Productive and Reproductive Performance of Nellore Heifers Grazing Mombasa Grass Pasture in Different Seasons

Abstract

The objective was to evaluate the effect of supplementation during three seasons (autumn, winter, and spring) on the productive and reproductive performance of Nellore heifers grazing on Mombaça grass. A total of 28 Nellore heifers were subjected to two supplementation strategies: 15 CP [15% crude protein (CP)] and 18 CP (18% CP). The experimental design was completely randomized in a factorial arrangement (two supplementation strategies and three seasons). A strategy × season interaction effect was observed for height (p = 0.008), with the 15 CP strategy in spring showing a higher average (56 cm) compared to 18 CP (26 cm). Regarding pasture chemical composition, the season significantly influenced quality (p < 0.05), with winter presenting inferior quality. A strategy × season interaction effect was also observed (p < 0.05), with lower averages recorded during winter for the 18 CP strategy in final body weight (FBW) (271.74 kg), total weight gain (TWG) (31.48 kg), and stocking rate (SR) (3.99 animal units per hectare, AU/ha). In reproductive efficiency, the 15 CP strategy achieved a pregnancy rate of 85% compared to 54% for the 18 CP strategy. The 15 CP strategy provided better performance for the heifers, increasing FBW, TWG, SR, and pregnancy rate, with significant results in spring due to improved forage quantity and quality associated with supplementation.

1. Introduction

Livestock production in tropical regions exhibits seasonal variations in forage quantity and quality, directly affecting animal performance [1]. During the rainy season, environmental conditions favor plant growth, leading to increased biomass production and higher forage nutritional value, which optimizes animal performance [2]. Conversely, in the dry season, there is an increase in NDF concentration, reducing forage intake and digestibility [3]. Simultaneously, the reduction in CP concentration in tropical pastures limits microbial growth, fiber digestion, and nutrient absorption, Ref. [4] compromising animal performance [5].

Sustainable pasture management is essential to maximize both animal productivity and pasture use efficiency [6]. In this context, the use of technologies such as concentrated supplementation is crucial, as it enhances livestock system productivity by improving individual performance, increasing stocking rates, achieving higher weight gains [7], and enhancing reproductive precocity [8].

Replacement heifers play a crucial role as potential future dams in beef cattle herds. However, this rearing phase is considered prolonged and costly, requiring significant investment. Therefore, promoting earlier age at first calving is essential to maximize the economic return of the livestock system, where grazing systems can help mitigate the costs associated with replacement heifers [9].

Nutrition is a key factor in achieving early pregnancy and ensuring proper body and reproductive development in heifers, especially during the months leading up to the breeding season [10]. Studies indicate that heifers subjected to protein supplementation tend to reach mature body weight earlier and achieve adequate physical conditions for reproduction [11,12,13].

Nutritional strategies should be implemented for grazing heifers, considering the possibility that pasture alone may not provide adequate nutritional balance [4], potentially leading to reduced animal performance [14]. Adequate protein supplementation enables higher rates of body weight gain [9] and prevents damage to mammary gland development in heifers [15].

However, diets with high concentrations of nitrogenous (above 18% crude protein–CP) compounds have been associated with reproductive developmental failures in female cattle due to excessive serum levels of ammonia and urea [16]. Conversely, Amundson et al. [17] did not observe deleterious effects on pregnancy rates in heifers fed supplements containing 14% CP.

Accordingly, the selection of the 15% CP level in the present study aimed to ensure a proper balance between protein supplementation and metabolic efficiency, while the 18% CP level was included to assess whether a higher protein intake would provide additional benefits to the productive and reproductive performance of heifers. The tested hypothesis was that 15% CP supplementation would be sufficient to meet the nutritional requirements of growing heifers without the potential adverse effects associated with excessive dietary protein. Furthermore, considering that forage quality may vary across seasons, the chosen levels allowed for an evaluation of whether protein supplementation could compensate for nutritional deficiencies in pasture, particularly during periods of lower forage nutritive value, such as those observed in winter.

Therefore, it is hypothesized that providing supplements with CP levels above 14% during the rearing phase of Nellore heifers may influence their productive and reproductive performance, either positively or negatively. Consequently, these strategies could have long-term implications for maintaining these parameters in future cows. This study aimed to evaluate two supplementation strategies during autumn, winter, and spring on the productive and reproductive performance of Nellore heifers.

2. Materials and Methods

2.1. Location, Treatments, and Animals

All procedures involving animals were approved by the Animal Use Ethics Committee, under protocol 013/2023. The experiment was conducted at Chácara Santa Luzia, located in the municipality of Araguaína, TO, Brazil, at 07°03′42″ S latitude and 48°13′26″ W longitude. The regional soil is classified as Orthic Quartzarenic Neosol [18].

The region’s climate is classified as AW4–Tropical with a wet summer and a dry winter, according to Köppen’s classification, with an annual precipitation of 1.900 mm [19]. Data on rainfall, relative humidity, and air temperature during the study period were obtained from the meteorological station at the Federal University of Northern Tocantins –Araguaína Campus (Figure 1).

A total of 28 Nellore heifers, approximately eight months old with an initial average body weight (BW) of 205.80 ± 16.50 kg, were used to evaluate performance and reproduction. The animals were allocated to a 4-hectare experimental area consisting of four 1.0-hectare paddocks, with pastures composed of Megathyrsus maximus cv. Mombasa, without tree coverage. For each strategy studied, two paddocks were assigned, with each paddock housing seven heifers, totaling 14 heifers per strategy. The paddocks were equipped with water troughs and covered feeding troughs accessible from both sides. A continuous grazing method with fixed stocking density was employed.

The experimental design was completely randomized in a factorial arrangement with two supplementation strategies (15 CP and 18 CP) and three periods (autumn, winter, and spring), with 14 replications (heifers). The evaluated strategies were as follows: 15 CP—Supplement containing 150 g/kg DM of Crude Protein (CP); and 18 CP—Supplement containing 180 g/kg DM of CP (

Table 1. Chemical composition of the concentrated supplements used (g/kg DM).

Variables	15 CP	18 CP
Dry matter (g/kg fresh matter)	888.49	892.58
Organic matter	933.53	929.86
Crude protein	151.26	179.56
Neutral detergent fiber	90.89	92.55
Acid detergent fiber	27.42	26.26
Total digestible nutrientes	867.64	868.54
Ether extract	36.86	37.51
Non fibrous carboydrates	740.99	801.83

). Supplements were supplied daily at 8:00 h. Before the experiment, all animals were identified, vaccinated against foot-and-mouth disease, and dewormed.

The supplementation was provided based on the forage availability during the evaluation periods (0.7%, 1.4%, and 0.9% for autumn, winter, and spring, respectively). The grazing cycles for the autumn and winter periods were 28 days each, while the spring cycles lasted 20 days. During the spring, with increased precipitation and rising temperatures (Figure 1), a single fertilization was carried out to ensure pasture development and keep the animals within the paddocks during the experiment. This procedure was necessary due to Mombasa grass’s high requirements for water, light, and nutrients [20]. An NPK fertilizer was used, with the formulated fertilizer (22-0-11) applied at rates of 33 kg/ha of nitrogen (N), 0 kg/ha of phosphorus (P), and 16 kg/ha of potassium (K).

2.2. Evaluation of Animal Performance

The heifers were weighed without prior fasting at the beginning and end of each grazing cycle to assess animal performance. The following variables were determined: Final dody weight (FBW, kg), total weight gain (TWG, kg = FBW − IBW), average daily gain (ADG, kg/day = TWG/days), stocking rate (SR, AU/ha = [((BW × number of animals)/450)/Area]), gain per area (GPA, @/ha = [(TWG × number of animals)/@]).

The dry matter intake (DMI) was estimated using the following Equation (1) for Zebu cattle from BR CORTE based on the ADG and body weight (BW) of each animal [21].

DMI_Estimated = −0.4684 + 0.7732 × DMI_Supplement + 0.0742 × BW^0.75 + 0.7953 × ADG − 0.9047 × ADG²

(1)

2.3. Evaluation of Forage Morphological Components

Samples for estimating the nutritive value and characterizing the forage availability were collected at representative points of the average height of the paddock. The canopy height was measured at the end of the grazing cycle, using a graduated ruler in centimeters, at 80 randomly selected points in each paddock. Four samples were collected from each paddock, 5 cm above the soil, using a rectangular frame (0.6 m²). After collection, the samples were transported to the laboratory and divided into two subsamples: one for estimating forage mass and the other separated into leaf blade, stem, and dead material [22].

Subsequently, the forage samples were dried in an oven at 55 ± 5 °C for 72 h and weighed to obtain the dry weight, in order to estimate the forage mass and determine the morphological components. The samples were then ground in a Willey-type knife mill (Marconi, Piracicaba, SP, Brazil) using a 1 mm sieve for further analysis.

2.4. Chemical Analyses

The samples of supplements and forage (whole plant) were quantified for dry matter (DM) (Method 976.01), ash (Method 942.05), crude protein (CP) (Method 968.06), and ether extract (EE) (Method 954.05) [23]. For the analysis of neutral detergent fiber corrected for ash and protein (NDF_CP) [24], neutral detergent fiber (NDF), and acid detergent fiber (ADF), these were determined according to Van Soest et al. [25], with adaptations by Detmann et al. [26], using an autoclave and the addition of thermostable α-amylase.

Due to the presence of urea in the supplements, the non-fibrous carbohydrate (NFC) content was determined using the following Equation (2) [27]:

NFC (%) = 100 − ((CP − CPu + U) + EE + ash + NDF)

(2)

To calculate the total carbohydrates (TC), Equation (3) determined by Sniffen et al. [28] was used:

TC (%) = 100 − (CP + EE + ash)

(3)

For the calculation of total digestible nutrients (TDN), two equations were used: the first Equation (4) for TDN of forage [29] and the second Equation (5) for TDN of the supplement [30]:

TDN_forage = 83.79 − (0.4171 × NDF (%) of forage)

(4)

TDN_supplement = 88.9 − (0.779 × AFD (%) of supplement)

(5)

2.5. Evaluation of the Fixed-Time Artificial Insemination Protocol

At the start of the reproductive protocol, heifers were required to reach 60% of the weight of an adult cow (450 kg), regardless of the season (autumn, winter, or spring). On the 160th day of the experiment, the heifers were induced to a hormonal protocol for estrus and ovulation synchronization to perform fixed-time artificial insemination (FTAI), using the methodology described by Prata et al. [31]. This protocol involves the induction of heifers with progesterone (P4) through the intramuscular administration of 1 mL of Sincrogest^® (Ouro Fino, Saúde Animal, Cravinhos, SP, Brazil) for 21 days.

On the day of induction, all heifers underwent a transrectal ultrasound examination (DP-2200 VET, Mindray, Shenzhen, China) to evaluate the reproductive tract score (RTS). The diameter of the uterine horns, the diameter of the largest follicle, and/or the presence of the corpus luteum were used as parameters in this evaluation, following the principles of the methodology described by Gutierrez et al. [32], with adaptations for local conditions (the order of the reproductive tract scores (RTS) and the uterine and ovarian characteristics described by Gutierrez et al. [32] were reversed to improve the understanding and practical application of the method). The evaluation used a score of 1 to 3, as shown in

Table 2. Description of reproductive tract scores (RTS) based on uterine and ovarian characteristics.

RTS	Uterine Horns Diameter	Ovarian Structure
1	≥15 mm	Corpus luteum present
2	≤15 mm	Follicles > 8.0 mm
3	<15 mm	Follicles < 8.0 mm

.

On the twenty-second day, considered day zero (D0) (Figure 2), a progesterone (P4) intravaginal device (Sincrogest^®, Ouro Fino, Saúde Animal, Cravinhos, SP, Brazil) was inserted, along with the intramuscular administration of 2 mL of estradiol benzoate (EB, Sincrodiol^®, Ouro Fino, Saúde Animal, Cravinhos, SP, Brazil). After seven days (D7), the P4 intravaginal devices were removed, with the removal time recorded to control the start of the activity within the parameters of the protocol.

All heifers received 1 mL of equine chorionic gonadotropin (eCG) (Ecegon^®, Biogénesis Bagó, Curitiba, Brazil), 2 mL of cloprostenol (Estron^®, Agener União, SP, Brazil), and 1 mL of estradiol cypionate (Croni-cip^®, Biogénesis Bagó, Curitiba, PR, Brazil) via intramuscular injection. Insemination was performed 48 h after implant removal, on day nine (D9). Pregnancy diagnosis was performed through transrectal ultrasonography, 40 days after insemination of the heifers. The pregnancy rate (PR) was determined by the ratio between the total number of pregnant heifers (PH) in a given strategy and the total number of eligible heifers (EH) in the FTAI within the same strategy, expressed as a percentage by the equation: PR = (PH/EH) × 100.

2.6. Statistical Analysis

The data were submitted to the Shapiro–Wilk test (p < 0.05), using PROC UNIVARIATE from SAS Institute. Once the assumptions of normality required for analysis of variance (ANOVA) were met, the MIXED procedure from SAS (SAS Institute, Inc., Cary, NC, USA) was used, with the evaluation periods included in the repeated measures model. Initial body weight was used as a covariate to adjust the other variables analyzed. When significant, the means between treatments were compared using Tukey’s minimum significant difference at a 5% probability level.

The data were analyzed as repeated measures over time using the following mathematical model:

Yijk = μ + Di + Tk + (DT)ik + εijk,

where

• Yijk is the observed value of the variable;
• μ is the overall mean effect;
• Di is the effect of the period;
• Tk is the effect of the treatment;
• (DT)ik is the effect of the interaction between the period and treatment;
• εijk is the experimental error effect.

The selection of the most appropriate covariance structure for the variation in the measurements within the treatment for each characteristic was carried out. This choice was based on the values of the Corrected Akaike Information Criterion (AICC) and the Bayesian Information Criterion (BIC), where the closer the value is to zero, the more suitable the matrix is, with the UN matrix being adopted.

3. Results

Throughout the experimental period, a drastic decrease in precipitation was observed in the month of August (Figure 1A), with the highest water deficit recorded during this month (Figure 1B). The average temperature remained relatively constant throughout the experimental period; however, it was noted that during the winter season, the minimum recorded temperatures were close to 18 °C (Figure 1).

For all productive and morphological components of the pasture, an effect of the strategy (p < 0.05) was observed (

Table 3. Productive and morphological components of the Megathyrsus maximus cv. Mombasa pasture throughout the grazing periods.

Variable	Strategy (S)		Period (P)			SEM	p-Value
	15 CP	18 CP	Autumn	Winter	Spring		S	P	S × P
Height, cm	47 A	26 B	45 a	23 b	41 a	1.926	0.001	<0.001	0.008
TFM, kg/ha	2.452 A	1.691 B	2.619 a	1.427 c	2.167 b	0.108	0.039	<0.001	0.446
GLM, kg/ha	1.549 A	0.919 B	1.311 b	0.642 c	1.750 a	0.086	0.019	<0.001	0.357
GLBM, kg/ha	0.577 A	0.390 B	0.421 b	0.122 c	0.907 a	0.053	0.002	<0.001	0.120
GSM, kg/ha	0.964 A	0.539 B	0.890 a	0.521 b	0.844 a	0.043	0.009	<0.001	0.805
DFM, kg/ha	1.134 A	0.812 B	1.446 a	0.918 b	0.555 c	0.064	0.006	<0.001	0.787
Leaf/Stem	0.82	0.74	0.56 b	0.39 c	1.40 a	0.076	0.485	<0.001	0.937

TFM, total forage mass; GLM, green forage mass; GLBM, green leaf blade mass; GSM, green stem mass; DFM, dead forage mass; SEM, standard error of the mean. Uppercase letters represent the strategy, and lowercase letters represent the period. Means followed by the same letters in the row do not differ from each other according to the Tukey test at 5% probability.

), except for the leaf/stem ratio (p = 0.485). The highest average values for these variables were observed for the 15 CP strategy.

For all the variables of productive and morphological components of the pasture, an effect of period was observed (p < 0.001) (Table 3). For total forage mass (TFM) and dead forage mass (DFM), the highest averages were observed during the autumn period (2.619 and 1.449 kg/ha, respectively). For green leaf mass (GLM) and green leaf blade mass (GLBM), higher averages were obtained in the spring period (1.750 and 0.907 kg/ha, respectively), and lower averages in the winter (0.642 and 0.122 kg/ha, respectively). For green stem mass (GSM), the highest averages were observed during autumn and spring (0.890 and 0.844 kg/ha, respectively). For leaf/stem ratio, a lower average was found in autumn (0.39), and a higher one in spring (1.40).

An effect of the interaction between strategy and period was observed only for height (p = 0.008) (

Table 4. Breakdown of the interaction of pasture height of Megathyrsus maximus cv. Mombasa across the grazing periods.

Variable	Strategy	Period			SEM	S vs. P
		Autumn	Winter	Spring
Height, cm	15 CP	55 a	29 b	56 Aa	1.926	0.008
	18 CP	36 a	16 c	26 Bb

SEM, standard error of the mean. Uppercase letters represent the strategy, and lowercase letters represent the cycle. Means followed by the same letters in the row do not differ from each other according to the Tukey test at 5% probability.

), where the highest values were found during the autumn and spring periods for the 15 CP strategy (55 and 56 cm, respectively).

For the chemical composition variables (

Table 5. Chemical composition of Megathyrsus maximus cv. Mombasa pasture throughout the grazing periods.

Variable	Strategy (S)		Period (P)			SEM	p-Value
	15 CP	18 CP	Autumn	Winter	Spring		S	P	S × P
DM, g/kg	386.60	397.80	287.80 b	514.90 a	374.00 b	2.249	0.746	<0.001	0.961
CP, g/kg	81.40	75.80	78.50 b	52.80 c	104.70 a	0.451	0.165	<0.001	0.466
NDF, g/kg	733.30	732.10	724.70 b	766.40 a	707.10 b	0.713	0.918	0.001	0.398
ADF, g/kg	407.70	404.20	392.40 b	447.30 a	378.10 b	0.872	0.792	<0.001	0.592

DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; SEM, standard error of the mean. Uppercase letters represent the strategy, and lowercase letters represent the period. Means followed by the same letters in the row do not differ from each other according to the Tukey test at 5% probability.

), no effects were observed for the interaction between strategy and period, nor for the individual strategy factor (p > 0.05). A period effect (p < 0.001) was observed for all chemical composition variables. For dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF), the highest averages were observed during the winter period (514.90, 766.40, and 447.30 g/kg, respectively). For crude protein (CP), the highest average was observed during the spring period (104.70 g/kg), and the lowest average during the winter period (52.80 g/kg).

An effect of strategy was observed for final body weight (FBW) (p = 0.012) and total weight gain (TWG) (p = 0.007), with the highest means recorded in the 15 CP strategy (

Table 6. Productive performance of Nellore heifers subjected to supplementation strategies for early calving.

Variable	Strategy (S)		Period (P)			SEM	p-Value
	15 CP	18 CP	Autumn	Winter	Spring		S	P	S × P
DMI, kg/day	6.14	5.98	4.97 b	6.91 a	6.49 a	0.101	0.747	<0.001	0.051
DMI, % BW	2.20	2.31	2.23	2.42	2.14	0.026	0.113	0.056	0.068
FBW, kg	337.83 A	328.38 B	241.36 c	277.83 b	333.11 a	4.667	0.012	<0.001	0.032
TWG, kg	131.52 A	123.05 B	35.54 b	36.47 b	55.29 a	1.337	0.007	<0.001	0.039
ADG, kg/day	0.677	0.639	0.635 b	0.434 c	0.907 a	0.025	0.129	<0.001	0.078
SR, AU/ha	4.14 A	4.04 B	3.48 c	4.04 b	4.75 a	0.065	0.017	<0.001	0.020
GPA, @/ha	10.24	9.55	8.29 b	8.51 b	12.90 a	0.312	0.077	<0.001	0.033

DMI, dry matter intake; BW, body weight; FBW, final body weight; TWG, total weight gain; ADG, average daily gain; SR, stocking rate; GPA, gain per area; SEM, standard error of the mean. Uppercase letters represent the strategies, and lowercase letters represent the periods. Means followed by the same letters in the row do not differ from each other according to the Tukey test at 5% probability.

). A period effect was observed for all performance variables (p < 0.001) except for dry matter intake (DMI, % BW) (p = 0.056). The highest means were observed during the spring period for DMI (kg/day), FBW, TWG, average daily gain (ADG), stocking rate, and gain per area (GPA). For ADG, it was observed that the lowest mean (0.434 kg/day) occurred during winter.

In the evaluation of animal performance (

Table 7. Breakdown of the interaction in the productive performance of Nellore heifers subjected to supplementation strategies for early calving.

Variable	Strategy	Period			SEM	S × P
		Autumn	Winter	Spring
FBW, kg	15 CP	242.62 c	283.91 Ab	337.83 Aa	4.667	0.032
	18 CP	240.09 c	271.74 Bb	328.38 Ba
TWG, kg	15 CP	36.31 b	41.46 Ab	54.09 a	1.337	0.039
	18 CP	34.76 b	31.48 Bb	56.48 a
SR, AU/ha	15 CP	3.49 c	4.09 Ab	4.83 a	0.065	0.020
	18 CP	3.47 c	3.99 Bb	4.67 a
GPA, @/ha	15 CP	8.46 b	9.66 Ab	12.61 a	0.312	0.033
	18 CP	8.12 b	7.35 Bb	13.19 a

FBW, final body weight; TWG, total weight gain; GPA, gain per area; SR, stocking rate; SEM, standard error of the mean. Uppercase letters represent the strategies, and lowercase letters represent the periods. Means followed by the same letters in the row do not differ from each other according to the Tukey test at 5% probability.

), an interaction effect of strategy x period was observed for FBW (p = 0.032), TWG (P = 0.039), stocking rate (SR, AU/ha) (p = 0.020), and gain per area (GPA, @/ha) (p = 0.033). During the winter period, the 18 CP strategy showed the lowest means for all the aforementioned variables, due to the seasonality of forage production, characterized by lower availability (Table 3) and nutritional quality (Table 5).

For the pregnancy rate results of the fixed-time artificial insemination protocol (FTAI), a rate of 85% was obtained for the 15 CP strategy and 54% for the 18 CP strategy, with the heifers having an average body weight of 278.15 kg and 279.46 kg for the 15 CP and 18 CP strategies, respectively.

4. Discussion

During the winter period, the lowest means were observed for all productive and morphological pasture component variables (Table 3), reflecting the water scarcity characteristic of this season (Figure 1B). It is worth highlighting that the water balance presented in Figure 1B is a critical element for understanding the seasonal variations in forage quality and availability during the experimental period. A more pronounced water deficit was observed during the winter months, characterized by low precipitation and high evapotranspiration. These factors negatively impacted the growth and renewal of grasses. This water scarcity reduced total forage mass, green leaf mass, and the leaf-to-stem ratio, leading to a decline in pasture nutritional quality, which was directly reflected in the productive and reproductive performance of the heifers. During periods of greater water availability, such as spring, the positive water balance favored grass regrowth, increasing the quantity of leaves available.

The seasonality of the pasture in winter is explained by the lower precipitation, solar radiation, temperature, and photoperiod, which alter the structure and quality of the pasture [33,34], consequently reducing forage growth and the appearance of new leaves. As a result, there is lower forage mass production [9].

The average total forage mass (TFM) for the 18 CP strategy (Table 3) was lower (1.691 kg/ha) than the value recommended by Minson [35], which is 2.000 kg/ha, to ensure selectivity and better animal performance. The morphological structure of the pasture and the nutritional value of the forage are determining factors for maximizing animal performance, as animals prefer leaves over stems, and the presence of stems in the grazing horizon limits the depth, area, and mass of the bite [36]. In this context, it was found that the amount of forage dry matter (FDM) was lower in winter (0.644 kg/ha) compared to other periods (1.311 and 1.750 kg/ha in autumn and spring, respectively), resulting in a smaller photosynthetic area and, consequently, delayed regrowth of the grass. Sbrissia et al. [37] emphasize that pasture management should ensure an adequate leaf area to stimulate rapid regrowth.

In the autumn season, a greater accumulation of dead forage mass (DFM) was observed (Table 3), a result in line with Ferreira et al. [34], who report that during the autumn/winter seasons, there is a higher production of DFM, attributed to low temperatures. The climatic conditions during this period were below (Figure 1A) the baseline temperature (15 °C) for tropical grasses, resulting in a reduction in the metabolic rate, which determines growth, and an increase in senescence [34]. Associated with environmental conditions, pasture management strategies can promote rapid leaf renewal, especially when combined with moderate grazing intensity under continuous stocking, due to the increased nitrogen (N) concentration in the forage mass [38]. These practices enhance N intake by grazing animals, resulting in greater N losses through excreta [39]. Therefore, the application of nitrogen fertilizers during the first rainfall events is crucial to ensure better pasture regrowth in tropical regions [40].

The chemical composition of the forage (Table 5) varied according to the evaluation periods, with these results attributed to the increased maturity of the leaves during the winter season [41], leading to lower nutritional value of the forage and a lower leaf/stem ratio (Table 3), which may explain the lower animal performance observed during winter (Table 6). During the autumn and spring periods, the pasture had sufficient CP (78.50 and 104.70 g/kg DM, respectively) (Table 5) to meet its nutritional requirements, enabling microbial growth and fiber digestion, as the protein concentration exceeded the range reported in the literature, which varies from 70 to 100 g/kg DM [4,35,42].

Nutritional strategies for grazing cattle aim to maximize or maintain desirable animal performance, particularly during forage production seasonality, where the goal is to improve the utilization of nutrients present in the pasture and stimulate pasture intake through supplementation. Silva et al. [43] reported that it is essential to understand the composition of the forage to make adjustments in supplement offerings, especially during transition periods.

In this study, variation in animal performance was observed throughout the periods (Table 6). Maximum performance was observed during the spring period, when there was a higher incidence of rainfall (Figure 1A), which favors grass regrowth. During the early stages of regrowth, leaves are the main morphological component in accumulated forage and the primary component consumed by the animal [44]. Another factor that may have contributed to the higher performance during this period is the amount of supplement being offered. Therefore, the animal’s response is dependent on the characteristics of the animals, environment, forage availability, and supplement offering [45].

During the winter, greater performance was observed for the 15 CP strategy compared to the 18 CP strategy (Table 7). These findings corroborate the observations of Gunter [46], which indicate that a higher supplement offering (% BW) can compensate for the decrease in both the quantity and quality of forage, resulting in the maintenance of productive gains, even with the increase in stocking rate. Supplementation plays a crucial role in intensifying grazing-based livestock systems by providing a greater quantity of nutrients to the animals [22], promoting an increase in stocking rate and greater gains per animal and per area [7].

The superior performance of the heifers receiving a supplementation level of 15 CP, compared to the 18 CP group, can be attributed to differences in the quality and quantity of the available forage. The heifers in the 15 CP group were managed on pastures with higher nutritional value, characterized by greater total forage mass, green forage mass, and leaf blade availability, particularly during the spring. These factors ensured a higher availability of leaves—the most nutritious part of the plant—thereby enhancing weight gain and reproductive performance. Furthermore, the 15 CP supplementation was sufficient to complement the nutrients present in the high-quality forage, promoting an efficient balance between protein and energy. In contrast, the excess protein in the 18 CP group was not fully utilized, leading to lower productive efficiency.

Supplementation increased the levels of CP in the diet, enhancing fiber intake, microbial growth, and fiber degradation [4]. Additionally, it contributed to greater N availability in animal metabolism [47]. These combined effects may have positively influenced the animal performance results observed in this study (Table 6). Lean et al. [16] report that diets with CP levels above 18% can impair the development of female cattle, primarily due to the excessive concentrations of ammonia and urea in serum.

The 15 CP strategy positively influenced the increase in pregnancy rate (85%), which was 31% higher compared to the pregnancy rate obtained with the 18 CP strategy. These results were influenced by the body weight (BW) achieved in this study, corresponding to 62% of the BW of an adult cow (450 kg). Pacheco et al. [48] and Silva et al. [49] state that Zebu heifers, when reaching 60% of the adult cow’s BW and subjected to reproductive protocols, present a pregnancy rate around 57%. Toledo et al. [50] highlight that the pursuit of reproductive precocity is more influenced by the heifer’s nutritional status than by age, with supplementation optimizing body development, fat deposition, and production of reproductive hormones, favoring progesterone synthesis, boosting luteal activity [51], and improving embryo quality [52].

Another important factor is the interaction between seasonality effects and pasture management. During the winter, both the quantity and quality of forage decreased in both groups (Table 3 and Table 5), due to the lack of alternative grazing areas during this period on the farm, particularly in the pre-reproductive phase, combined with adverse climatic conditions (high temperatures, low precipitation, and soil water deficit) (Figure 1A,B). These factors may have contributed to the lower performance and pregnancy rates observed in the 18 CP strategy, as reported in the study by Silva et al. [49]. Consequently, this led to a reduction in the quality of the diet consumed by the animals, despite the provision of supplementation. Studies by Thatcher and Collier [53] and Rocha et al. [54] demonstrate that elevated temperatures can disrupt the balance between estrogen and progesterone, leading to embryonic mortality prior to implantation or embryonic underdevelopment. This effect is aggravated by reduced uterine blood flow and increased uterine temperature, significantly compromising embryonic viability [49,55]. Additionally, the high protein levels provided to the 18 CP group may have increased serum levels of urea and ammonia, impairing reproductive performance and potentially explaining the lower pregnancy rate (54%) compared to the 85% observed in the 15 CP group. These results highlight the importance of balancing protein supplementation with the quality of available forage to optimize animal performance and reproductive indices. Several studies indicate that the long-term impact of protein supplementation in heifers is a maximization of muscle development in the progeny [56,57,58]. However, it is important to note that the small sample size was one of the limitations of this study. Therefore, further research is needed to validate these findings.

Based on the results obtained in this study, the analysis of the productive and morphological components of the pasture demonstrates seasonal variations throughout the year, forming a strategic integration with supplementation to address the specific challenges of each period. Among the morphological components that have the greatest effect on animals are green leaves, which are influenced by photoperiod, temperature, light, nutrition, and precipitation, directly impacting the development of tropical grasses [59]. According to Roth et al. [60], the structural characteristics of the forage directly influence animal consumption during grazing and, consequently, their performance.

Additionally, supplementation plays a crucial role in the production of early-maturing heifers, enhancing significant gains during the different evaluated seasons. Nutritional status plays a major role in the onset of puberty, and in this context, body weight is a factor that directly affects reproductive precocity. Thus, supplementation is a strategy employed to promote better animal performance and accelerate reproductive precocity [61]. Supplementation showed substantial impacts on animal performance, playing a key role in optimizing pregnancy rate.

5. Conclusions

Among the supplementation strategies evaluated, the 15 CP strategy provided better performance for heifers on pasture, resulting in increased final body weight gain, total weight gain, and stocking rate, as well as improved reproductive efficiency with a higher pregnancy rate. Additionally, during the spring period, the animals showed better performance due to the improvement in the quality of available forage, supported by the adoption of sustainable management practices, using technologies that enhance input efficiency, along with supplementation.