Evaluation of Forage Yield and Quality of Double-Cut Persian Clover (Trifolium resupinatum) Lines under Weather Conditions of Karaj, Iran

Document Type : RESEARCH ARTICLE

Authors

1 . Associate Professor, Maize and Forage Crops Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.

2 Ph. D. in Agronomy, Maize and Forage Crops Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.

3 Assistant Professor, Maize and Forage Crops Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran.

10.22092/spj.2026.373580.1475

Abstract

To evaluate forage yield and quality of double-cut Persian clover lines and to identify superior lines, a field experiment was carried-out in 2023-24 and 2024-25 growing seasons at the research farm of the Seed and Plant Improvement Institute, Karaj, Iran. The experiment was arranged as split-plot in time in randomized complete block design with three replications. Sixteen Persian clover lines were evaluated in two cuts. The measured and studied traits were dry forage yield, crude protein content and yield, acid detergent fiber (ADF), neutral detergent fiber (NDF), digestible dry matter content and yield, water-soluble carbohydrates, ash content, dry matter intake, relative feed value and net energy for lactation. Combined analysis of variance revealed significant year × line and year × cut interactions for dry forage yield, crude protein content and yield, digestible dry matter content and yield, acid detergent fiber, water-soluble carbohydrates, ash content, and net energy for lactation. Line 9 (BPC/Lordegan) consistently outperformed the other lines, particularly in the second growing season, producing the highest dry forage yield (3.28 t ha-1), crude protein content (25.8%), crude protein yield (843 kg ha-1), and digestible dry matter yield (2144 kg ha-1). In addition, Lines 10 (BPC/Qourshibashi), 13 (KPC/2chin14), 12 (KPC/2chin13), 8 (BPC/Kermanshah), and 5 (BPC/Shazand) ranked among the superior lines based on both forage yield and nutritive value. Overall, Lines 9 and 10 demonstrated superior forage yield and nutritional quality in the two growing seasons. These two lines were identified suitable for release as new cultivars for agroecological conditions similar to those of Karaj as well as potential parents in Persian clover breeding programs.
 
Keywords: Perian clover, crude protein, dry forage yield, dry matter intake, forage digestibility.
 
Introduction
Persian clover (Trifolium resupinatum) is one of the most important annual forage legumes grown in Mediterranean and semi-arid regions because of its high forage quality, rapid establishment, excellent palatability, and ability to biologically fix atmospheric nitrogen (Bakhtiyari et al., 2020). The productivity and nutritional value of forage legumes are controlled by both genetic and environmental conditions, and significant genotype × environment interactions often influence their agronomic performance (Zamanian et al., 2025a).
Previous studies have reported considerable genetic variation among Persian clover populations for forage yield, crude protein content, fiber fractions, and digestibility (Zamanian et al., 2024a). Nevertheless, information regarding double-cut Persian clover germplasm, particularly its forage yield and quality, and regrowth performance under temperate conditions of Iran, remains scarce.
Regrowth ability directly determines total seasonal forage production, identifying superior double-cut Persian clover genotypes is essential for development of improved cultivars with high productivity and nutritional value. Therefore, the objective of this study was to evaluate 16 double-cut Persian clover lines under the environmental conditions of Karaj in two successive growing seasons, to determine their forage yield and nutritional value, and to identify promising breeding lines suitable for release as new cultivars and sustainable forage production.
 
Materials and methods
The experiment was carried-out in 2023-24 and 2024-25 growing seasons at the research farm of the Seed and Plant Improvement Institute, Karaj, Iran. Sixteen double-cut Persian clover lines originating from different regions of Iran were evaluated using split-plot in time arrangement in randomized complete block design with three replications, where harvest time (first and second cut) was considered the subplot factor. Before sowing, soil samples (0-30 cm depth) were collected for physio-chemical properties analysis.
 Considering the resuts of soil analysis, 150 kg ha-1 triple superphosphate and 50 kg ha-1 urea were incorporated into the soil as basal fertilizers. Seeds with 99% germinability were sown manually on 19 September 2023 and 22 September 2024 in a depth of 3 cm in the soil. Each plot consisted of four 5-m length rows with 50 cm row spacing. Irrigation was applied immediately after sowing and subsequently at 7–10 day intervals according to crop water requirements.
Forage was cut in late April (at approximately 25% flowering), and the second cut was taken after regrowth reached the same phenological stage. Border effects were eliminated, and forage was harvested from the central 5 m2 area of each plot. For determination of dry forage yield and forage quality, samples were oven-dried at 60°C for 72 hours to reach to constant weight. Dried samples were ground using a laboratory mill and passed through a fine sieve before chemical analysis.
Crude protein content (CPC), water-soluble carbohydrates (WSC), ash, acid detergent fiber (ADF), neutral detergent fiber (NDF), and digestible dry matter (DDM) were determined using Near Infrared Reflectance Spectroscopy. Dry matter intake (DMI), relative feed value (RFV), net energy for lactation (NEL), crude protein yield (CPY), and digestible dry matter yield (DDMY) were calculated using standard equations (Zamanian et al., 2024a).
Experimental data were subjected to combined analysis of variance using SAS 9.1. Year was considered as a random effect, and line and cut were considered as fixed effects. Mean comparisons were performed using the least significant difference (LSD) test at the 5% probability level.
 
Results and Discussion
Combined analysis of variance revealed significant year × line and year × cut interactions for dry forage yield, CPC, CPY, ADF, DDM, DDMY, WSC, ash content, and NEL, indicating considerable genetic diversity and differential responses of Persian clover lines to environmental conditions and successive forage harvests. These findings confirm that both genotype and seasonal weather conditions play important roles in determining forage productivity and nutritional value (Zamanian et al., 2025a).
The more favorable weather conditions during the second growing season significantly increased dry forage yield, CPC, CPY, DDMY, WSC, and NEL, while reducing differences in quality between the first and second cuts. This result indicates that favorable weather conditions enhanced regrowth ability and allowed plants to maintain forage quality after the first harvest. Among all evaluated lines, Line 9 (BPC/Lordegan) consistently exhibited superior agronomic and nutritional value performance in both growing seasons.
In the second growing season, Line 9 produced the highest dry forage yield (3.28 t ha-1), CPC (25.8%), CPY (843 kg ha-1), and DDMY (2144 kg ha-1). Furthermore, it had the lowest ADF and NDF concentrations together with the highest digestibility, DMI, RFV, and NEL. These characteristics indicate that Line 9 combined high forage productivity with excellent nutritional value. Lines 10 (BPC/Qourshibashi), 13 (KPC/2chin14), 12 (KPC/2chin13), 8 (BPC/Kermanshah), and 5 (BPC/Shazand) also ranked among the superior lines for both forage yield and quality. Their consistently favorable performance in two growing seasons suggests their high adaptability and breeding value for improving Persian clover germplasm (Zamanian et al., 2025a).
While several lines experienced reductions in forage quality during the second cut, Lines 9 and 10 maintained relatively high CPC, digestibility, WSC, and RFV. Such stability in quality parameters indicates superior genetic and physiological capacity for regrowth and efficient redistribution of assimilates following the first harvest (Pourali et al., 2021).
Overall, the results of the present study demonstrated that considerable genetic variability exists among double-cut Persian clover germplasm. Simultaneous improvement of forage yield and nutritional value appears feasible, because the best-performing lines combined high biomass production with desirable fiber composition and digestibility (Zamanian et al., 2024a). Consequently, Lines 9 and 10 were identified as promising lines for release as new cultivars as well as valuable parents in future Persian clover breeding programs targeting temperate and semi-arid environments, similar to Karaj, in Iran.
 
References
Bakhtiyari, F., Zamanian, M. and Golzardi, F. 2020. Effect of mixed intercropping of clover on forage yield and quality. South-Western Journal of Horticulture, Biology and Environment, 11(1), pp.49–65.
Pourali, S., Aghayari, F., Ardakani, M.R., Paknejad, F. and Golzardi, F. 2023. Benefits from intercropped forage sorghum–red clover under drought stress conditions. Gesunde Pflanzen, 75, pp.1769–1780. DOI: 10.1007/s10343-023-00833-4
Zamanian, M., Golzardi, F., Gitari, H., Nungula, E., Salehi, F. and Heydarzadeh, S. 2024a. Enhancing forage nutritional value in Persian clover (Trifolium resupinatum) and crimson clover (Trifolium incarnatum) through intercropping and optimized seeding rate. Cogent Food and Agriculture, 10(1), e2410459. DOI: 10.1080/23311932.2024.2410459
Zamanian, M., Heydarzadeh, S. and Golzardi, F. 2025a. Evaluation of forage yield and quality of single-cut Persian clover (Trifolium resupinatum) lines under climatic conditions of Karaj in Iran. Seed and Plant, 40, pp.241–270 (in Persian). DOI: 10.22092/spj.2025.369383.1416

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References

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Seed and Plant Journal
Volume 42, Issue 1
April 2026
Pages 1-36

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