Introduction

The selection and management of replacement cows in tropical herds is one of the most important factors affecting livestock development in these areas. Nutritional and reproductive constraints associated with lower weight gain after weaning are more marked than in animals raised in temperate climates (Mukassa-Mugerwa 1989).

In a recent study, Ahmed and El-Hag (2004) evaluated the nutritional content of several species of grass in tropical conditions and suggested that during the dry season, the pastures did not support the nutritional requirements of maintenance of the animals. Protein supplementation increase microbial activity, ruminal digestion, and growth improvement of heifers (Aranda et al. 2001) with no significant changes in body condition score (Slanac et al. 2007).

Previous studies in the humid tropics in Costa Rica showed that increasing protein amount in the supplement given to heifers (5.5% to 13% crude protein) increased the proportion of animals with a corpus luteum (CL), but had no effect on weight gain and body condition score (BCS; Maquivar et al. 2006). Heifers raised in the tropics are subjected to seasonal variations in forage quality and availability (Aranda et al. 2001); during the dry season, grass growth vegetation is impaired which causes tissue mobilization during this time of the year. In periods of abundance, body weight gain is masked by enhanced gut fill, whereas body weight increments do not reflect changes in adiposity and lean tissue deposition (NRC 2001). Therefore, the use of the body condition score (Flamenbaum et al. 1995) in combination with back fat thickness (BFT) using measurement by real-time ultrasound (Mösenfetchel et al. 2002) could be a good indicator in the assessment of the energetic condition in replacement heifers. Consequently, the objectives of this study were to evaluate the effects of protein supplementation on the productive and reproductive performance of heifers in the tropics.

Materials and methods

Location

The study was conducted at the experimental station of the Technological Institute of Costa Rica, located in San Ramón, Alajuela (10°25′ N and 84°32′ W) at 172 m above sea level. The region is classified as tropical, with a mean annual temperature of 27.3°C and rainfall 3,062 mm, with a relative humidity of 85.3%.

Animals and treatments

Forty-five heifers (Bos taurus × Bos indicus), 673 ± 146 days of age, were divided into two treatments. The supplemented group (SG; n = 22) with an initial body weight (BW) of 340.7 ± 34.4 kg received a concentrate at 1% ratio of the average BW (13% crude protein (CP) and 3.15 Mcal/kg of digestible energy; Citrocarne® Casa Dos Pinos, Costa Rica). The control treatments were not supplemented (NSG; n = 23) and had an initial BW of 339.7 ± 39.35 kg. Animals were adapted to the concentrate over a 15-day period and thereafter supplemented for 30 days. Heifers were fed as a group in a confined area and were grazing a mixture of pastures based on African stargrass (Cynodon nlemfuensis), Ratana (Ischaemum indicum), and Candelario (Pennisetum purpureum) with free access water and mineral salts (Ganafos Plus® Piensos S.A., Costa Rica).

Forage analysis

Grass samples, clipped at approximately 10 cm above ground level, were collected at the beginning of each grazing period in five 0.5-m2 plots for chemical analysis. Nitrogen content was determined by macro-Kjeldahl technique (AOAC 1990), neutral detergent fiber (NDF), and acid detergent fiber (ADF) using the procedures outlined by Van Soest et al. (1991). Rumen degradable protein (RDP) was estimated by analyzing the residual nitrogen of the in situ incubation of the supplement in polyester bags (Ahmed and El-Hag 2004), the indigestible protein as N in ADF (Van Soest et al. 1991), and escape protein (EP) was estimated by difference between these two fractions. Forage composition was 86.6% dry matter (DM), 10.7% CP, 57.8% NDF, and 40.1% ADF. The supplement composition was 92.5% DM, 15.9% CP, 7.2% RDP, EP 4.7%, and 3.6% indigestible protein.

Ultrasonography measurements

Heifers were weighed every 15 days and BCS recorded on a scale from 1 to 5, where 1 = emaciated and 5 = obese (Edmonson et al. 1989). At the same time, animals were scanned with an Aloka SSD 500-V real-time scanner (Tokyo, Japan) using a 7.5-MHz sectorial probe to measure BFT. The hair at each measurement point was clipped close to the skin, and a gel was used as a coupling medium. The probes were placed perpendicular to the backbone between the third and fourth lumbar vertebrae (Silva et al. 2005). Within treatments, animals were classified into two groups for analysis: heifers with an average BFT equal or superior to 0.7 cm and those with a BFT of less than 0.69 cm; therefore, four groups were compared: supplemented heifers with high BFT (SGH), supplemented heifers with low BFT (SGL), nonsupplemented heifers with high BFT (NSGH), and nonsupplemented heifers with low BFT (NSGL).

Dry matter intake

Five animals of the supplemented group were dosed intraruminally with 3 g of chromic oxide (Cr2O3) for 15 days (Aranda et al. 2001). Fecal grab samples (200 g/day) were collected during the last 7 days of this period as recommended. Feed and fecal samples were oven dried (60°C, 48 h) and ground (1 mm screen) and composited by animal. Samples for chromium analysis were determined by atomic absorption spectroscopy. Dry herbage intake and digestibility were calculated with internal markers as described by Aranda et al. (2001).

Estrous synchronization procedure

After the dietary supplementation period, estrus was synchronized using a progesterone implant (Norgestomet, Crestar®, Lab. Intervet, Mexico), placed in the ear for 9 days. An intramuscular injection of estradiol valerate was given at the time of implant insertion. After implant withdrawal, estrus was detected continuously for 56 h. Heifers were inseminated 12 h after the onset of estrus. Blood samples were collected at the same time as ultrasonographic evaluations by venipuncture of the coccygeal vein or artery. Samples were centrifuged at 7,000 rpm for 20 min, and serum progesterone was analyzed by solid phase radioimmunoassay (Pulido et al. 1991). Heifers with a CL were defined by two consecutive values of progesterone above 1 ng/ml (Zalesky et al. 1984).

Statistical analyses

Ovulation and pregnancy rates were analyzed by chi-square test. Dry matter intake (DMI) and average of BCS were compared with a Student’s t test. Average daily weight gain and BFT were analyzed using a completely randomized design with initial weight as a covariable (Steel and Torrie 1996).

Results and discussion

The supplemented group had a better average daily weight gain than the control group (0.63 kg ± 0.16 vs. 0.51 kg ± 0.13, P < 0.05) without any differences seen between treatments in the average BW. No differences were observed at the end of the experiment in DMI, BW, nor in BCS. No differences were observed between groups regarding overall initial BFT measurements and final BFT measurement (Table 1). The BFT differences within treatments were found at day 30 and at day 45 (Fig. 1). No difference was observed between the percentages of animals that were cycling before the beginning of the experiment (23% for both treatments). Overall, supplemented females tended to show a better percentage of ovulation at the end of the experiment than those in the nonsupplemented group (P < 0.10), but this event was not reflected in the final pregnancy rates (Table 1).

Table 1 Effect of protein supplementation on productive parameters and reproductive performance in heifers raised in the tropics of Costa Rica
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Fig. 1
figure 1

Body fat thcikness (BFT) from initiation of supplementation (day 0) to day 45 on diet. Solid line (suplemented group), dashed line (nonsupplemented group). Different letters indicates statistical difference within treatments (P < 0.05)

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The BFT classification analysis showed significant differences among groups (Fig. 2). Heifers from the supplemented group with a high BFT showed a better ovulation percentage than those with low BFT (P < 0.01). This categorization also showed that the pregnancy rate of supplemented heifers with high BFT tended to be better in comparison with heifers with low BFT in this same group (67% vs. 30%, respectively, P < 0.10). In contrast, no differences were observed between BFT categories in the nonsupplemented group (Table 2). A larger sample size might have helped to clarify the advantage of supplementation on pregnancy rates.

Fig. 2
figure 2

Body fat thickness from initiation of supplementation (D0) to day 45 on diet. Regrouped of heifers according with the BFT measurements, heifers were classified in four groups: SGH (supplemented animals with an average equal or superior to 0.7 cm) and SGL (supplemented females with less than 0.69 cm), NSGH (nonsupplemented animals with an average equal or superior to 0.7 cm) and NSGL (nonsupplemented females with less than 0.69 cm). ab P < 0.05; c P < 0.10

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Table 2 Effects of protein supplementation on body fat thickness classification and percentage of ovulation and pregnancy in heifers raised in the tropics of Costa Rica
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Even when there was a better daily gain in the supplemented group, this was relatively low due to the poor content of EP in the supplement (4.79%). Some studies have shown that increasing the amount of dietary CP in B. indicus accelerates the onset of puberty (Fajersson et al. 1991). Oyedipe et al. (1982) reduced the time to the onset of puberty from 23 months with 8.3% CP to 18 months with 19.1% CP. In the present study, heifers were 2 years of age at the beginning of the experiment; therefore, no relationship was established between supplementation and onset of puberty; however, it was observed that heifers with thicker back fat tissue and also supplemented showed higher rates of ovulation and higher conception percentage that animals from supplemented treatment with lower BFT and nonsupplemented treatment with either lower or higher BFT. Thus, monitoring BFT appears to be a valuable tool to select animals requiring supplementation, but this statement requires further investigation.

In some studies, DMI has been increased with protein supplementation in animals raised under tropical pastures, particularly when the protein content of the forage is lower than 6% CP (Aranda et al. 2001). However, in the present experiment, since pastures contained 10.8% CP, there was probably no response to rumen degradable protein supplementation on DMI. Thus, in order to observe differences in DMI, it is likely that the quality of the pasture given should be lower in protein content. Notwithstanding, in two previous studies carried out at the same experimental station in Costa Rica, intake was not affected in heifers supplemented with low or medium protein content concentrates (5.5% CP or 13% CP; Maquivar et al. 2006). The effects of the interaction between protein content of the forage and the supplement provided merits further investigation. It is important under the conditions of the tropics to determine if pastures are nitrogen deficient, in order to decide on the use of protein supplementation. This insufficiency may affect nutrient intake and result in a negative effect on fertility, growth, and reproductive performance.

Results from this study indicate that protein supplementation in tropical forages improved average daily gain and tended to enhance the proportion of animals that ovulated in a synchronization program; nonetheless, the differences can be attributed to the characteristics of the forage and the supplement. No differences were observed between treatments in BCS, BW, and overall BFT, but supplemented animals with high BFT showed better reproductive performance than those with low BFT. These data suggest that adipose tissue may play a role in the reproductive process in addition to its dietary effect.

There is increasing interest in the use of real-time ultrasound as a noninvasive and cost-effective method to measure body composition in replacement beef cattle (Mösenfetchel et al. 2002). However, perhaps due to the small differences in weight gain, changes in overall body fat thickness were scarcely detected. It is well known that reproductive performance in cattle is affected by mobilization of body energy reserves, particularly during the period near the onset of puberty and the one that followed parturition (Lalman et al. 1997).

In the present study, some animals receiving the supplement had high BFT and showed better reproductive performance, while others did not. More information is needed to understand these differences. Protein supplementation improved the reproductive performance, and additionally, use of ultrasonography to measure the back fat thickness represents a reliable tool to estimate the reproductive performance of the animals. Further studies, however, are needed to establish the importance of monitoring back fat and its relationship with the reproductive performance, as well as to assess the economic impact and management advantage of this technique for supplemented heifers in humid tropical environments.