Preparation of sweet lupin seeds

The raw seed was roasted using a pan for 15 minutes at a temperature of 130 ^oC and then cooled and dehulled using a roller mill and exposed to the air for completed separation of the cotyledons from hulls. Hammer mill was used to grind the roasted seed as described by Laudadio and Tufarelli (2011)Laudadio, V. & Tufarelli, V. (2011). Influence of substituting dietary soybean meal for dehulled-micronized lupin (Lupinus albus cv. Multitalia) on early phase laying hens’ production and egg quality. Livestock Science, 140(1-3): 184-188, ISSN: 1878-0490. https://doi.org/10.1016/j.livsci.2011.03.029. to produce the meal which is referred hereafter as heat-treated dehulled lupin (HDL).

Research design and birds’ management

Total of 160 Lohmann pre-lay stage pullets of 16 weeks age was obtained from the Alema Farms PLC (Bishoftu, Ethiopia). They were kept at the experimental site until they start laying eggs during which they were fed with pre-layer commercial ration. The actual experiment commenced when the pullets were 19 weeks age. Four dietary treatments were formulated to contain 0 % HDL (HDL0), 9 % HDL (HDL9), 18 % HDL (HDL18) and 27 % HDL (HDL27) (table 1). The hens were then randomly distributed to each treatment diets replicated four times containing 10 chicks each. The treatment diets were formulated to be isocaloric and isonitrogenous with similar energy density and protein levels (table 2).

The experimental pens were cleaned, disinfected, and watering and feeding equipment were cleaned and disinfected as well. The birds were kept in 1.5 m x 1.65m wire mesh partitioned pens and the concrete floor was covered with sawdust of about 5 cm deep. The wet litter was replaced with dried and cleaned sawdust whenever necessary. Sunlight was used as a source of natural light during the day period, and additional supplementary fluorescent light were used at evening for a total of about 16 hours. Water was provided ad libitum. A 20 % amprolium powder solution (12 g per 20 litres) was administered through drinking water for 5 to 7 days as a prophylactic measure against coccidiosis.

Data collection procedures

Data collection on egg production commenced when the birds were 22 weeks old and continued until they reached 37 weeks of age, spanning 16 consecutive weeks. The birds were provided with their respective dietary treatments twice daily: once in the morning at 08:00 and again in the afternoon at 16:00. The daily weighed allowance was divided into two equal portions. Feed refusal was collected the following day before the next feeding is offered. Feed intake was calculated by subtracting the feed refusal from the offered feed.

Eggs were collected twice a day from each pen at 08:00 and 17:00. As there was no mortality recorded during the experimental period, egg production was determined on a hen-housed basis, considering the number of hens initially housed. The weight of each egg was recorded daily and averaged. Total egg mass output was computed by multiplying the total number of eggs produced during the experimental period with the average weight of these eggs. The feed conversion ratio (kg feed/kg egg mass) was then calculated by dividing the total feed consumed by the total egg mass produced.

The egg quality characteristics were measured at four age points. The first, second, third and fourth egg quality characteristics were determined at the ages of 24, 28, and 32 and 36 weeks, respectively. Eggs were collected for five consecutive days at each age point and stored in a cool room. Individual eggs and dry shell weight were measured using a triple beam balance. The egg length, egg width, and yolk diameter were measured with a digital calliper. A tripod micrometre gauge was used to measure the heights of albumen and yolk. The yolk colour was determined using the Rosche Yolk Colour Fan. The Haugh unit (HU) score was calculated according to Eisen et al. (1962)Eisen, E.J., Bohren, B.B. & McKean, H.E. (1962). The Haugh unit as a measure of egg albumen quality. Poultry Science, 41: 1461-1468, ISSN: 1525-3171. https://doi.org/10.3382/ps.0411461. by applying the average albumen height and egg weight into the equation: HU = 100log (albumen height + 7.57 − 1.7 * egg weight^0.37). The yolk index was calculated by dividing the yolk height by its width.

The egg dimensions, length (L) and width (W) were used to compute the following geometrical parameters: Egg shape index was calculated as the ratio of W to L multiplied by 100. The surface area of the egg was computed using the method of Narushin et al. (2021a)Narushin, V.G., Romanov, M.N. & Griffin, D.K. (2021a). Non-destructive measurement of chicken egg characteristics: Improved formulae for calculating egg volume and surface area. Biosystems Engineering, 201: 42-49, ISSN: 1537-5129. https://doi.org/10.1016/j.biosystemseng.2020.11.006. as 0.933W (W+2.343L). The proportion of shell was determined by dividing the dried shell weight by the egg weight. The eggshell density was calculated as follows:

Chemical composition analysis

Samples of the soybean meal, the heat-treated dehulled lupin seed meal and feeds offered were analysed for dry matter, ash, ether extract, and crude fiber using the procedures of AOAC (1995)AOAC. (1995). Official Methods of Analysis the Association of Official Analytical Chemists, 16^th edn., Arlington, VA, USA. at the Animal Nutrition Laboratory of Hawassa University. The crude protein, calcium, and phosphorus were analysed at the Holeta Agricultural Research Centre (Ethiopia). The crude protein content was analysed using the micro-Kjeldahl method while the concentrations of calcium and phosphorous were determined by atomic absorption spectrophotometer (dry ashing) as described by AOAC (1995)AOAC. (1995). Official Methods of Analysis the Association of Official Analytical Chemists, 16^th edn., Arlington, VA, USA. . The metabolizable energy of diets was estimated based on the feed composition tables of tropical feeds for poultry, while that of heat-treated dehulled lupin was computed using the equation of Wiseman (1987)Wiseman, J. (1987). Meeting nutritional requirements from available resources. In: J. Wiseman (Ed). Feeding of Non-Ruminant Animals. Butterworth and C. Ltd. London, UK. pp. 132..

Statistical Analysis

Data on feed intake, egg production, egg weight, egg mass and feed conversion ratio were analysed using one-way ANOVA by considering the effects of treatment diets as a single fixed factor. Since the interaction effects of treatment by age was not significant for all egg quality characteristics, it was dropped from the statistical analysis. Accordingly, the data for egg qualities measured at four age points were pooled and subjected to one-way ANOVA by fitting treatment diets as a single factor effect. The significance of mean differences was analysed using the post-hoc Tukey test. To check the age effect, data on treatment diets for egg qualities were further pooled and subjected to General Linear procedure (due to missing values) by fitting age as a single factor. The Tukey-Kramer test for adjusted means was then applied to compare the least square means of the age effect. Additionally, the orthogonal polynomial contrast trend analysis was conducted to determine the linear and quadratic effects on increasing levels of the dietary lupin substitutions. All statistical analysis was performed using the Statistical Analysis System (SAS 2016, ver. 9.4Statistical Analysis System (SAS). (2016). Statistical Analysis System, Ver. 9.4, SAS Institute Inc., Cary, NC, USA.) and results are presented in least square means along with pooled standard error of the mean.

Feed intake and egg production

As presented in table 2, the fat content and metabolizable energy concentration in HDL was comparable to that of soybean meal (12.9 % in HDL vs. 12.5 % in soybean meal and 3316 kcal/kg in HDL vs. 3500 kcal/kg in soybean meal). As shown in table 3, the mean values for total egg number, egg weight and egg mass were similar (P>0.05) across dietary treatments which accords with the reports of Lee et al. (2016a)Lee, M.R., Parkinson, S., Fleming, H.R., Theobald, V.J., Leemans, D.K. & Burgess, T. (2016a). The potential of blue lupins as a protein source, in the diets of laying hens. Veterinary and Animal Science, 1: 29-35, ISSN: 2451-943X. https://doi.org/10.1016/j.vas.2016.11.004. . Furthermore, the findings align with those of Struti et al. (2021)Struti, D.I., Bunea, A., Pop, I.M., Păpuc, T.A. & Mierliţă, D.P. (2021). The influence of dehulling on the nutritional quality of lupine seeds (Lupinus albus L.) and the effect of their use in the feed of laying quails on the live performance and quality of eggs. Animals, 11(10): 2898, ISSN: 2076-2615. https://doi.org/10.3390/Struţi/ani11102898. , who reported that incorporating dehulled lupin seeds into the diet of quails did not affect egg production compared to the control group. Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. reported that a 10-20 % inclusion of lupin seeds with a 10 % inclusion of peas had no negative effect on most egg production traits. However, they reported that the highest dose of narrow-leaved lupin (25 %) in the diet was associated with reduced weight of eggs.

The egg mass has linearly decreased (P=0.03) with increased substitution levels of soybean meal with HDL the highest reduction being observed at 27 %. Egg mass is the product of total egg number and average egg weight, and the observed reduction might be associated with low levels of essential amino acids in the lupin seed. Researchers have revealed that most lupin species exhibit a significant deficiency in sulphur-containing amino acids, primarily methionine, which is essential for the synthesis of cysteine and phenylalanine (Roman et al. 2023Roman, L., Tsochatzis, E., Tarin, K., Röndahl, E.M., Ottosen, C.O. & Corredig, M. (2023). Compositional attributes of blue lupin (Lupinus angustifolius) seeds for selection of high-protein cultivars. Journal of Agricultural and Food Chemistry, 71(45): 17308-17320, ISSN: 1520-5118. https://doi.org/10.1021/acs.jafc.3c04804. and Kponouglo et al. 2024Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. ).

On the other hand, diets containing graded levels of heat-treated dehulled lupin seed meal had affected the total and average daily feed intake of laying hens. Consequently, the substitution of soybean meal at 27 % of heat-treated dehulled lupin seed meal has resulted in a decrease (P<0.039) of feed intake in comparison to the remaining treatments. Similarly, total feed intake linearly decreased across treatment groups (P=0.044). None of the variables showed a significant trend of a quadratic effect. Consistent with the current results, Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. observed depressed feed intake and poor feed conversion ratio in Hy-Line Brown hens fed diet with 25 % yellow lupin seeds. The reduction in feed consumption may be attributed to the presence of alkaloids and oligosaccharides in lupine seeds because high levels of alkaloids can markedly reduce protein digestibility (Jeroch et al. 2016Jeroch, H., Kozłowski, K., Schöne, F. & Zduńczyk, Z. (2016). Lupines (Lupinus spp.) as a protein feedstuff for poultry. 1) Varieties, composition and nutritional values for poultry. European Poultry Science, 80(125), ISSN: 1612-9199. http://dx.doi.org/10.1399/eps.2016.125. ). Moreover, Pham et al. (2020)Pham, H.D., Siddik, M.A., Fotedar, R., Chaklader, M.R., Foysal, M.J., Nguyen, C.M. & Munilkumar, S. (2020). Substituting fishmeal with lupin Lupinus angustifolius kernel meal in the diets of cobia Rachycentron canadum: Effects on growth performance, nutrient utilization, haemato-physiological response, and intestinal health. Animal Feed Science and Technology, 267: 114556, ISSN: 1873-2216. https://doi.org/10.1016/j.anifeedsci.2020.114556. reported that L. angustifolius seeds contained higher levels of structural carbohydrates, which may have negatively influenced the feed consumption of the hens.

On the other hand, Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. reported that the increased levels of non-processed lupin seeds in the diets of laying hens had positively influenced the average daily feed intake. They revealed that the daily feed intake of birds fed with lupin diets has increased at week six as compared with the control group. In another study, the substitution of soybean meal at 7, 10, and 15 % for dehulled lupin seed meal improved egg production and egg weight output per hen as compared to control diet (Andrianova et al. 2019Andrianova, E.N., Egorov, I.A., Grigor'eva, E.N., Shevyakov, A.N. & Pronin, V.V. (2019). Lupine is applicable in diets for layer chicken of parental flock. Agricultural Biology, 54(2): 326-336, ISSN: 2412-0324. https://doi.org/10.15389/agrobiology.2019.2.326eng. ). The differences reported by varies researchers on the effect of lupin seed on feed consumption might be associated with the seed variety, physical activity of the chickens, types of housing system used and the environmental conditions in which the birdy were raised.

Egg quality characteristics

As shown in table 4, egg width, dry shell weight, shell ratio and shell density were affected (P<0.05) by treatments diets being higher in hens fed with HDL0 diet than that of HDL27. No significant differences were observed between chickens fed with HDL0, HDL9, and HDL18 diets in terms of the same characteristics. However, egg width was significantly higher (P<0.05) in hens fed with the HDL0 diet than those fed the HDL18 and HDL27 diets. A linear decrease (P<0.015) was observed in shell weight, eggshell ratio, and eggshell density across the treatment diets.

In the current study, egg shape index was not affected by treatment diets which disagrees with the observations of Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. who reported that the egg shape index in narrow-leaved lupin fed group was higher than the control group. The observed reduction in the dry shell weight in chickens fed with treatment diets is in line with that of Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. who reported a deterioration trend of egg quality traits with the increased levels of non-processed yellow lupin (Lupinus luteus L.) inclusion in poultry diets. They observed that a 10-25 % inclusion of lupin reduced the proportion of eggshell in each nutritional group. Eggshell is made almost entirely of calcium carbonate (CaCO₃) crystals. The supply of adequate amounts of mineral calcium in layer hens’ diet is thus essential. The calcium content in heat-treated dehulled lupin was lower than that of soybean meal and this may be attributed to the dehulling process, as calcium is predominantly deposited in the hull, resulting in reduced concentration after dehulling (Park et al. 2016Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. ). The observed egg shape index score agrees with the findings of Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. who reported comparative values for hens fed fermented lupin seed. In the poultry industry, egg shape index is considered as one of the important characteristics for uniformed packages of eggs during transportation and delivery by decreasing the potential of breakage eggs. An egg shape index of 75 is regarded as the most satisfactory which accords to the present findings.

The quality of albumen and yolk is of particular interest to the consumers of poultry products (Tolimir et al. 2017Tolimir, N., Maslovaric, M., Skrbic, Z., Lukic, M., Rajkovic, B. & Radisic R. (2017). Consumer criteria for purchasing eggs and the quality of eggs in the markets of the city of Belgrade. Biotechnology in Animal Husbandry, 33(4): 425-437, ISSN: 2217-7140. https://doi.org/10.2298/BAH1704425T. ). The quality of both albumen and yolk is expressed by proportion and index (Kraus et al. 2019Kraus, A., Zita, L. & Krunt, O. (2019). The effect of different housing system on quality parameters of eggs in relationship to the age in brown egg-laying hens. Bulgarian Journal of Agricultural Science, 25(6): 1246-1253, ISSN: 2534-983X. http://www.agrojournal.org. and 2021Kraus, A., Zita, L., Krunt, O., Härtlová, H. & Chmelíková, E. (2021). Determination of selected biochemical parameters in blood serum and egg quality of Czech and Slovak native hens depending on the housing system and hen age. Poultry Science, 100(2): 1142-1153, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2020.10.039. ). Haugh unit scores are a crucial parameter for assessing albumen quality, which helps to determine the overall quality of egg content and its freshness (Narushin et al. 2021bNarushin, V.G., Romanov, M.N. & Griffin, D.K. (2021b). A novel egg quality index as an alternative to Haugh unit score. Journal of Food Engineering, 289: 110176, ISSN: 1873-5770. https://doi.org/10.1016/j.jfoodeng.2020.110176. ). As presented in table 5, no significant difference was observed between treatment diets in all internal egg quality characteristics which is consistent with the observations of Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. and Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. . Similarly, the orthogonal polynomial contrast analysis did not show any linear or quadratic effects on treatment diets for internal qualities. Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. reported that a 15 % inclusion of lupin was associated with a higher proportion of thin albumen in eggs. However, the current study did not reveal any significant differences in the albumen quality between treatment diets. In contrast to the current study, Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. reported that in narrow-leaved lupin fed group the height of thick albumen, Haugh score, and yolk colour was higher than the control group. Such differences might be due to the variety of lupin used by various researchers.

Consistent with the current findings, Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. and Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. reported that diets containing narrow-leaf and fermented lupin seed had no effect on the albumen height and Haugh unit scores. In another study, Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. reported higher Haugh unit scores than observed in the current study for brown laying hens fed unprocessed different levels of lupin seed (Lupinus angustifolius L.) supplementation. Although not significant, yolk colour linearly increased with increased treatment diets which is in accordance with the findings of Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. who reported that supplementation of unprocessed lupin seed supplementation in laying hen diets linearly improved the yolk colour. Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. also revealed a significant improvement of yolk colour in the group fed diet containing 25 % yellow lupin seeds (Lupinus luteus L.).

Inclusion of blue lupin seed meals in the diets of layer hens has been reported to improve the yolk fat profiles which is essential for the health of consumers (Kowalska et al. 2020Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. ). Research conducted by Timová et al. (2020)Timová, I., Straková, E., Všetičková, L. & Suchý, P. (2020). Impact of feeding mixture containing lupin meal on improvement of polyunsaturated fatty acids in egg yolk. Czech Journal of Animal Science, 65(08): 311-321, ISSN: 1805-9309. https://doi.org/10.17221/87/2020-CJAS. demonstrated that replacing extracted soybean with white lupin seed meal in feed mixtures, at both 50 % and 100 % levels as a source of protein, significantly decreased the saturated fatty acids and increased the mono- and polyunsaturated fatty acids, which are essential for consumers’ health, especially for prevention of coronary diseases. The cholesterol levels of eggs produced from hens fed with fermented lupin seed (Lupinus angustifolius) was significantly lower than those fed on the control diet (Kponouglo et al. 2024Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. ). Similarly, Lim and Choi (2023)Lim, C.I. & Choi, N.J. (2023). Effect of lupin (Lupinus angustifolius) as a soybean meal replacement on the performance, meat quality, and blood parameters of broilers. Canadian Journal of Animal Science, 103(2): 167-173, ISSN: 1918-1825. https://dx.doi.org/10.1139/CJAS-2022-0104. observed significantly higher levels of polyunsaturated fatty acids in broiler chickens fed with 10 % and 20 % lupin seed (Lupinus angustifolius) meal than in those fed the control diet.

Age effect on egg quality

As presented in table 6, dry shell weight and yolk colour were not affected by age while egg weight, egg length, egg width, yolk height and width, albumen height and Haugh unit score significantly and linearly increased with age. These observations are in line with those reported by Yurtseven et al. (2021)Yurtseven, E.P., Şekeroğlu, A., Tainika, B., Duman, M. & Şentürk, Y.E. (2021). Effect of production system and age on egg quality parameters: A case of Niğde Province Çamardı District, Turkey. Turkish Journal of Agriculture-Food Science and Technology, 9(8): 1407-1412, ISSN: 2148-127X. https://doi.org/10.24925/turjaf.v9i8.1407-1412.4241. for chickens reared in the free-range production system. Similarly, Manyeula et al. (2021)Manyeula, F., Sebolai, B., Sempule, G. & Moreki, J.C. (2021). Effects of broiler breeders’ Age on egg quality characteristics and their correlation coefficients. Journal of World's Poultry Research, 11(3): 368-375, ISSN: 2322-455X. https://dx.doi.org/10.36380/jwpr.2021.44. reported that egg weight, egg length, egg width, egg yolk, egg content, egg volume, shell percentage, albumen weight, and egg surface area increased with the age of the hen. On the other hand, indices of egg shape and yolk significantly decreased with the advanced hens’ age and these observations are in line with those reported by Kraus et al. (2021)Kraus, A., Zita, L., Krunt, O., Härtlová, H. & Chmelíková, E. (2021). Determination of selected biochemical parameters in blood serum and egg quality of Czech and Slovak native hens depending on the housing system and hen age. Poultry Science, 100(2): 1142-1153, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2020.10.039. and Tainika et al. (2024)Tainika, B., Şekeroğlu, A., Akyol, A., Şentürk, Y.E., Abaci, S.H. & Duman, M. (2024). Effects of age, housing environment, and strain on physical egg quality parameters of laying hens. Brazilian Journal of Poultry Science, 26(03): 001-014, ISSN: 1806-9061. eRBCA-2024. http://dx.doi.org/10.1590/1806-9061-2024-1911. . The latter authors reported a significant decline of shell breaking strength with the age of then hens while shell thickness showed inconsistent pattern. In another study, Lee et al. (2016b)Lee, M.H., Cho, E.J., Choi, E.S., Bang, M.H. & Sohn, S.H. (2016b). The effect of hen age on egg quality in commercial layer. Korean Journal of Poultry Science, 43(4): 253-261, ISSN: 2287-5387. http://dx.doi.org/10.5536/KJPS.2016.43.4.253. , Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. and Sam (2023)Sam, I.M. (2023). The influence of Isa Brown laying hen age on egg quality in humid tropics. Animal Research International, 20(2): 4966-4972, ISSN: 1597-3115. https://oaji.net. reported a significant decline in albumen height, Haugh unit score, and yolk color with increasing hens’ age.

An increase of egg shape index with the age of the hen has been reported by Manyeula et al. (2021)Manyeula, F., Sebolai, B., Sempule, G. & Moreki, J.C. (2021). Effects of broiler breeders’ Age on egg quality characteristics and their correlation coefficients. Journal of World's Poultry Research, 11(3): 368-375, ISSN: 2322-455X. https://dx.doi.org/10.36380/jwpr.2021.44. which contrasts with the present observation. Such variations might be due to the chicken breed and type of diet used and the age of the hens when the data were collected. For instance, Lee et al. (2016b)Lee, M.H., Cho, E.J., Choi, E.S., Bang, M.H. & Sohn, S.H. (2016b). The effect of hen age on egg quality in commercial layer. Korean Journal of Poultry Science, 43(4): 253-261, ISSN: 2287-5387. http://dx.doi.org/10.5536/KJPS.2016.43.4.253. and Alo et al. (2024)Alo, E.T., Daramola, J.O., Wheto M. & Oke, O.E. (2024). Impact of broiler breeder hens’ age and egg storage on egg quality, embryonic development, and hatching traits of FUNAAB-alpha chickens. Poultry Science, 103: 103313, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2023.103313. have revealed that eggs from 60-week-old hens had lower albumen height, and Haugh unit score values compared to eggs from 30-week-old hens. Chung and Lee (2014)Chung, S.H. & Lee, K.W. (2014). Effect of hen age, storage duration and temperature on egg quality in laying hens. International Journal of Poultry Science, 13: 634-636, ISSN: 1994-7992. https://doi.org/10.3923/ijps.2014.634.636. reported that egg quality was not affected by the age of the hen. In contrary, Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. found a significant difference in egg quality characteristics between age of hens. Egg weight, shell density, shell weight, breaking strength and thickness significantly increased with the age of hens (from 19 to 39 weeks of age). The same authors reported a significant decline in height at albumen and Haugh unit score which disagrees with the results of the current study.

The Haugh unit score has been widely accepted as the “gold standard” for quantifying the internal quality and freshness of eggs. The Haugh unit score quickly declines over time, making it a sensitive measure of egg deterioration soon after being laid. On the other hand, the yolk index quantifies the structural integrity of the egg yolk, which gradually decreases over the storage period enabling the identification of quality differences among eggs that have deteriorated over time (Yurtseven et al. 2021Yurtseven, E.P., Şekeroğlu, A., Tainika, B., Duman, M. & Şentürk, Y.E. (2021). Effect of production system and age on egg quality parameters: A case of Niğde Province Çamardı District, Turkey. Turkish Journal of Agriculture-Food Science and Technology, 9(8): 1407-1412, ISSN: 2148-127X. https://doi.org/10.24925/turjaf.v9i8.1407-1412.4241. ). In contrast, the effect of age on yolk colour in the current study was found to be insignificant. Yolk colour is affected by the diet of the birds mainly by the presence of xanthophyll resulting in variations in the depth of the yolk colour (Lordelo et al. 2017Lordelo, M., Fernandes, E., Bessa, R.J.B. & Alves, S.P. (2017). Quality of eggs from different laying hen production systems, from indigenous breeds and specialty eggs. Poultry Science, 96(5): 1485-1491, ISSN: 1525-3171. http://dx.doi.org/10.3382/ps/pew409. ).

Preparación de semillas dulces de lupino

La semilla cruda se tostó en una sartén durante 15 minutos a una temperatura de 130 °C y luego se enfrió y se descascaró utilizando un molino de rodillos, exponiéndola al aire para lograr la separación completa de los cotiledones de las cáscaras. Se utilizó un molino de martillos para triturar la semilla tostada, tal como lo describen Laudadio y Tufarelli (2011)Laudadio, V. & Tufarelli, V. (2011). Influence of substituting dietary soybean meal for dehulled-micronized lupin (Lupinus albus cv. Multitalia) on early phase laying hens’ production and egg quality. Livestock Science, 140(1-3): 184-188, ISSN: 1878-0490. https://doi.org/10.1016/j.livsci.2011.03.029. , para producir la harina que en lo sucesivo se denominará lupino descascarado tratado con calor (HDL).

Diseño de la investigación y manejo de las aves

Se obtuvieron un total de 160 pollitas Lohmann en etapa pre-postura de 16 semanas de edad de Alema Farms PLC (Bishoftu, Etiopía). Se mantuvieron en el sitio experimental hasta que comenzaron a poner huevos, durante el cual se les alimentó con una ración comercial para pre-postura. El experimento comenzó cuando las pollitas tenían 19 semanas de edad. Se formularon cuatro tratamientos dietéticos para contener 0 % HDL (HDL0), 9 % HDL (HDL9), 18 % HDL (HDL18) y 27 % HDL (HDL27) (tabla 1). Luego, las gallinas se distribuyeron aleatoriamente por cada tratamiento y se replicó cuatro veces con 10 pollitas cada una. Las dietas de tratamiento se formularon para ser isocalóricas e isoproteicas, con similar densidad energética y niveles de proteína (tabla 2).

Los corrales experimentales, así como los bebederos y comederos, se limpiaron y desinfectaron. Las aves se mantuvieron en corrales de malla metálica de 1.5 m x 1.65 m, divididos en compartimentos, y el piso de concreto estaba cubierto con aserrín de unos 5 cm de profundidad. La cama húmeda se reemplazaba con aserrín seco y limpio siempre que fuera necesario. Durante el día se utilizaba la luz solar como fuente de iluminación natural, y por la noche se añadía luz fluorescente complementaria para un total de aproximadamente 16 horas. Se proporcionaba agua ad libitum. Se administró una solución de polvo de amprolio al 20 % (12 g por 20 litros) a través del agua de bebida durante 5 a 7 días como medida profiláctica contra la coccidiosis.

Procedimientos de recolección de datos

La recolección de datos sobre la producción de huevos comenzó cuando las aves tenían 22 semanas de edad y continuó hasta que alcanzaron las 37 semanas, abarcando 16 semanas consecutivas. A las aves se les proporcionaron sus tratamientos dietéticos respectivos dos veces al día: una vez por la mañana a las 08:00 y nuevamente por la tarde a las 16:00. La ración diaria pesada se dividió en dos porciones iguales. El rechazo de alimento se recogió al día siguiente antes de ofrecer la siguiente alimentación. El consumo de alimento se calculó restando el rechazo de alimento del alimento ofrecido. Los huevos se recogieron dos veces al día de cada corral a las 08:00 y a las 17:00. Como no se registró mortalidad durante el período experimental, la producción de huevos se determinó de acuerdo con las gallinas alojadas, considerando el número inicial de gallinas. El peso de cada huevo se registró diariamente y se promedió. La producción total de masa de huevo se calculó multiplicando el número total de huevos producidos durante el período experimental por el peso promedio de estos huevos. La relación de conversión alimenticia (kg de alimento/kg de masa de huevo) se calculó entonces dividiendo el total de alimento consumido entre la masa total de huevos producida.

Las características de calidad del huevo se midieron en cuatro puntos de edad. Las primeras, segundas, terceras y cuartas características de calidad del huevo se determinaron a las edades de 24, 28, 32 y 36 semanas, respectivamente. Los huevos se recogieron durante cinco días consecutivos en cada punto de edad y se almacenaron en una sala fría. Los huevos individuales y el peso de la cáscara seca se midieron utilizando una balanza de tres brazos. La longitud del huevo, el ancho del huevo y el diámetro de la yema se midieron con un calibrador digital. Se utilizó un micrómetro de trípode para medir las alturas del albumen y la yema. El color de la yema se determinó utilizando el abanico de color de yema de Rosche. La puntuación de la unidad Haugh (UH) se calculó según Eisen et al. (1962)Eisen, E.J., Bohren, B.B. & McKean, H.E. (1962). The Haugh unit as a measure of egg albumen quality. Poultry Science, 41: 1461-1468, ISSN: 1525-3171. https://doi.org/10.3382/ps.0411461. , aplicando la altura promedio del albumen y el peso del huevo en la ecuación: UH = 100log (altura del albumen + 7.57 - 1.7 * peso del huevo^0.37). El índice de yema se calculó dividiendo la altura de la yema por su ancho.

Se utilizaron las dimensiones del huevo, longitud (L) y ancho (W), para calcular los siguientes parámetros geométricos: índice de forma del huevo, que se calculó como la relación de W a L multiplicada por 100. El área de la superficie del huevo se calculó utilizando el método de Narushin et al. (2021a)Narushin, V.G., Romanov, M.N. & Griffin, D.K. (2021a). Non-destructive measurement of chicken egg characteristics: Improved formulae for calculating egg volume and surface area. Biosystems Engineering, 201: 42-49, ISSN: 1537-5129. https://doi.org/10.1016/j.biosystemseng.2020.11.006. como 0.933W (W+2.343L). La proporción de cáscara se determinó dividiendo el peso de la cáscara seca entre el peso del huevo. La densidad de la cáscara del huevo se calculó de la siguiente manera:

Análisis de composición química

Se analizaron muestras de harina de soya, harina de semillas de lupino descascaradas y tratadas con calor, y los alimentos ofrecidos para determinar materia seca, cenizas, extracto etéreo y fibra cruda utilizando los procedimientos de la AOAC (1995)AOAC. (1995). Official Methods of Analysis the Association of Official Analytical Chemists, 16^th edn., Arlington, VA, USA. en el Laboratorio de Nutrición Animal de la Universidad de Hawassa. La proteína cruda, calcio y fósforo se analizaron en el Centro de Investigación Agrícola de Holeta (Etiopía). El contenido de proteína cruda se analizó mediante el método micro-Kjeldahl, mientras que las concentraciones de calcio y fósforo se determinaron mediante espectrofotometría de absorción atómica (incineración en seco) según lo descrito por la AOAC (1995)AOAC. (1995). Official Methods of Analysis the Association of Official Analytical Chemists, 16^th edn., Arlington, VA, USA. . La energía metabolizable de las dietas se estimó con base en las tablas de composición de alimentos tropicales para aves, mientras que la de los lupinos descascarados y tratados con calor se calculó usando la ecuación de Wiseman (1987)Wiseman, J. (1987). Meeting nutritional requirements from available resources. In: J. Wiseman (Ed). Feeding of Non-Ruminant Animals. Butterworth and C. Ltd. London, UK. pp. 132..

Análisis Estadístico

Los datos sobre consumo de alimento, producción de huevos, peso del huevo, masa de huevo y la relación de conversión alimenticia se analizaron utilizando ANOVA simple, considerando los efectos de las dietas de tratamiento como un único factor fijo. Dado que los efectos de interacción del tratamiento por edad no fueron significativos para todas las características de calidad del huevo, se excluyeron del análisis estadístico. En consecuencia, los datos de calidad de huevo medidos en cuatro puntos de edad se agruparon y se sometieron a ANOVA simple, considerando las dietas de tratamiento como único efecto factorial. La significancia de las diferencias entre medias se analizó utilizando la prueba post-hoc de Tukey. Para comprobar el efecto de la edad, los datos sobre las dietas de tratamiento para la calidad de huevo se agruparon adicionalmente y se sometieron al procedimiento Lineal General (debido a valores faltantes) considerando la edad como único factor. Posteriormente, se aplicó la prueba de Tukey-Kramer para medias ajustadas para comparar las medias cuadráticas del efecto de la edad. Además, se realizó un análisis de tendencias mediante contrastes de polinomios ortogonales para determinar los efectos lineales y cuadráticos de los niveles crecientes de sustituciones dietéticas de lupino. Todos los análisis estadísticos se realizaron con el Sistema de Análisis Estadístico (SAS 2016, ver. 9.4Statistical Analysis System (SAS). (2016). Statistical Analysis System, Ver. 9.4, SAS Institute Inc., Cary, NC, USA.) y los resultados se presentan en medias cuadráticas junto con el error estándar promedio combinado.

Consumo de alimento y producción de huevos

Como se presenta en la tabla 2, el contenido de grasa y la concentración de energía metabolizable en HDL fueron similares a los de la harina de soya (12.9 % en HDL ante 12.5 % en harina de soya y 3316 kcal/kg en HDL frente a 3500 kcal/kg en harina de soya). Como se muestra en la tabla 3, los valores promedio del número total de huevos, el peso del huevo y la masa de huevo fueron similares (P>0.05) entre los tratamientos dietéticos, lo cual coincide con los informes de Lee et al. (2016a)Lee, M.R., Parkinson, S., Fleming, H.R., Theobald, V.J., Leemans, D.K. & Burgess, T. (2016a). The potential of blue lupins as a protein source, in the diets of laying hens. Veterinary and Animal Science, 1: 29-35, ISSN: 2451-943X. https://doi.org/10.1016/j.vas.2016.11.004. . Además, los hallazgos se alinean con los de Struti et al. (2021)Struti, D.I., Bunea, A., Pop, I.M., Păpuc, T.A. & Mierliţă, D.P. (2021). The influence of dehulling on the nutritional quality of lupine seeds (Lupinus albus L.) and the effect of their use in the feed of laying quails on the live performance and quality of eggs. Animals, 11(10): 2898, ISSN: 2076-2615. https://doi.org/10.3390/Struţi/ani11102898. , quienes reportaron que la incorporación de semillas de lupino descascaradas en la dieta de codornices no afectó la producción de huevos en comparación con el grupo control. Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. reportaron que una inclusión del 10 al 20 % de semillas de lupino junto con una inclusión del 10 % de guisantes no tuvo un efecto negativo en la mayoría de las características de producción de huevos. Sin embargo, reportaron que la dosis más alta de lupino de hoja estrecha (25 %) en la dieta se asoció con una reducción en el peso de los huevos.

La masa de huevo ha disminuido linealmente (P=0.03) con niveles crecientes de sustitución de harina de soya por HDL, observándose la mayor reducción al 27 %. La masa de huevo es el producto del número total de huevos y el peso promedio de los huevos, y la reducción observada podría estar asociada a bajos niveles de aminoácidos esenciales en la semilla de lupino. Los investigadores han revelado que la mayoría de las especies de lupino presentan una deficiencia significativa en aminoácidos que contienen azufre, principalmente metionina, que es esencial para la síntesis de cisteína y fenilalanina (Roman et al. 2023Roman, L., Tsochatzis, E., Tarin, K., Röndahl, E.M., Ottosen, C.O. & Corredig, M. (2023). Compositional attributes of blue lupin (Lupinus angustifolius) seeds for selection of high-protein cultivars. Journal of Agricultural and Food Chemistry, 71(45): 17308-17320, ISSN: 1520-5118. https://doi.org/10.1021/acs.jafc.3c04804. y Kponouglo et al. 2024Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. ).

Por otro lado, las dietas que contenían niveles mayores de harina de semillas de lupino descascaradas y tratadas con calor afectaron el consumo total y diario medio de alimento de las gallinas ponedoras. En consecuencia, la sustitución de harina de soja por un 27 % de harina de semillas de lupino descascaradas y tratadas con calor resultó en una disminución (P<0.039) del consumo de alimento en comparación con los demás tratamientos. De igual manera, el consumo total de alimento disminuyó linealmente entre los grupos de tratamiento (P=0.044). Ninguna de las variables mostró una tendencia significativa de efecto cuadrático. Como los resultados actuales, Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. observaron disminución del consumo de alimento y una relación de conversión del alimento deficiente en gallinas Hy-Line Brown, alimentadas con una dieta que contenía 25 % de semillas de lupino amarillo. La reducción en el consumo de alimento puede atribuirse a la presencia de alcaloides y oligosacáridos en las semillas de lupino, ya que niveles elevados de alcaloides pueden reducir notablemente la digestibilidad de proteínas (Jeroch et al. 2016Jeroch, H., Kozłowski, K., Schöne, F. & Zduńczyk, Z. (2016). Lupines (Lupinus spp.) as a protein feedstuff for poultry. 1) Varieties, composition and nutritional values for poultry. European Poultry Science, 80(125), ISSN: 1612-9199. http://dx.doi.org/10.1399/eps.2016.125. ). Además, Pham et al. (2020)Pham, H.D., Siddik, M.A., Fotedar, R., Chaklader, M.R., Foysal, M.J., Nguyen, C.M. & Munilkumar, S. (2020). Substituting fishmeal with lupin Lupinus angustifolius kernel meal in the diets of cobia Rachycentron canadum: Effects on growth performance, nutrient utilization, haemato-physiological response, and intestinal health. Animal Feed Science and Technology, 267: 114556, ISSN: 1873-2216. https://doi.org/10.1016/j.anifeedsci.2020.114556. informaron que las semillas de L. angustifolius contenían niveles más altos de carbohidratos estructurales, lo que podría haber tenido una influencia negativa en el consumo de alimento de las gallinas.

Por otro lado, Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. informaron que altos niveles de semillas de lupino sin procesar en la dieta de gallinas ponedoras influyó positivamente en la ingesta diaria promedio de alimento. Revelaron que el consumo diario de alimento de las aves alimentadas con dietas de lupino aumentó en la semana seis en comparación con el grupo de control. En otro estudio, la sustitución de harina de soja en un 7, 10 y 15 % por harina de semilla descascarada de lupino mejoró la producción de huevos y el peso de los huevos por gallina en comparación con la dieta de control (Andrianova et al. 2019Andrianova, E.N., Egorov, I.A., Grigor'eva, E.N., Shevyakov, A.N. & Pronin, V.V. (2019). Lupine is applicable in diets for layer chicken of parental flock. Agricultural Biology, 54(2): 326-336, ISSN: 2412-0324. https://doi.org/10.15389/agrobiology.2019.2.326eng. ). Las diferencias que reportaron diversos investigadores sobre el efecto de la semilla de lupino en el consumo de alimento podrían estar asociadas con la variedad de la semilla, la actividad física de las aves, los tipos de sistemas de alojamiento utilizados y las condiciones ambientales en las que se criaron las aves.

Características de calidad del huevo

Como se muestra en la tabla 4, el ancho del huevo, el peso seco de la cáscara, la proporción de cáscara y la densidad de la cáscara se afectaron (P<0.05) por las dietas de tratamiento, siendo mayores en las gallinas alimentadas con la dieta HDL0 que en las de HDL27. No se observaron diferencias significativas entre las gallinas alimentadas con las dietas HDL0, HDL9 y HDL18 en cuanto a las mismas características. Sin embargo, el ancho del huevo fue significativamente mayor (P<0.05) en las gallinas alimentadas con la dieta HDL0 que en aquellas alimentadas con las dietas HDL18 y HDL27. Se observó una disminución lineal (P<0.015) en el peso de la cáscara, la proporción de la cáscara y la densidad de la cáscara en las dietas de tratamiento.

En el estudio actual, el índice de forma del huevo no se vio afectado por las dietas de tratamiento, lo cual no concuerda con las observaciones de Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. , quienes reportaron que el índice de forma del huevo en el grupo alimentado con lupino de hojas estrechas fue mayor que en el grupo control. La reducción observada en el peso seco de la cáscara en pollos alimentados con dietas de tratamiento coincide con los hallazgos de Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. , quienes reportaron una tendencia de deterioro en las características de calidad del huevo con niveles crecientes de inclusión de lupino amarillo (Lupinus luteus L.) no procesado en dietas para aves de corral. Observaron que una inclusión de 10-25 % de lupino redujo la proporción de cáscara de huevo en cada grupo nutricional. La cáscara de huevo está compuesta casi en su totalidad por cristales de carbonato de calcio (CaCO₃). Por lo tanto, el suministro de cantidades adecuadas de calcio mineral en la dieta de las gallinas ponedoras es esencial. El contenido de calcio en el lupino descascarado y tratado con calor fue menor que en la harina de soja y esto puede atribuirse al proceso de descascarado, ya que el calcio se deposita predominantemente en la cáscara, lo que resulta en una menor concentración después de eliminada esta (Park et al. 2016Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. ). El puntaje observado del índice de forma del huevo concuerda con los hallazgos de Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. , quienes reportaron valores comparativos para gallinas alimentadas con semillas de lupino fermentadas. En la industria avícola, el índice de forma del huevo se considera una de las características importantes para paquetes uniformes de huevos durante el transporte y entrega, al disminuir la probabilidad de rotura de los huevos. Un índice de forma del huevo de 75 se considera el más satisfactorio, lo que coincide con la presente investigación.

La calidad de la clara y la yema es de particular interés para los consumidores de productos avícolas (Tolimir et al. 2017Tolimir, N., Maslovaric, M., Skrbic, Z., Lukic, M., Rajkovic, B. & Radisic R. (2017). Consumer criteria for purchasing eggs and the quality of eggs in the markets of the city of Belgrade. Biotechnology in Animal Husbandry, 33(4): 425-437, ISSN: 2217-7140. https://doi.org/10.2298/BAH1704425T. ). La calidad de la clara y la yema se expresa mediante proporción e índice (Kraus et al. 2019Kraus, A., Zita, L. & Krunt, O. (2019). The effect of different housing system on quality parameters of eggs in relationship to the age in brown egg-laying hens. Bulgarian Journal of Agricultural Science, 25(6): 1246-1253, ISSN: 2534-983X. http://www.agrojournal.org. y 2021Kraus, A., Zita, L., Krunt, O., Härtlová, H. & Chmelíková, E. (2021). Determination of selected biochemical parameters in blood serum and egg quality of Czech and Slovak native hens depending on the housing system and hen age. Poultry Science, 100(2): 1142-1153, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2020.10.039. ). Los valores de la unidad Haugh son un parámetro crucial para evaluar la calidad de la clara, lo que ayuda a determinar la calidad general del contenido del huevo y su frescura (Narushin et al. 2021bNarushin, V.G., Romanov, M.N. & Griffin, D.K. (2021b). A novel egg quality index as an alternative to Haugh unit score. Journal of Food Engineering, 289: 110176, ISSN: 1873-5770. https://doi.org/10.1016/j.jfoodeng.2020.110176. ). Como se presenta en la tabla 5, no se observaron diferencias significativas entre las dietas de tratamiento en todas las características internas de calidad del huevo, lo cual concuerda con las observaciones de Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. y Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. . De manera similar, el análisis de contraste polinomial ortogonal no mostró ningún efecto lineal o cuadrático de las dietas de tratamiento en las cualidades internas. Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. informaron que la inclusión del 15 % de lupino se asoció con una mayor proporción de clara delgada en los huevos. Sin embargo, el presente estudio no reveló diferencias significativas en la calidad de la clara entre las dietas de tratamiento. En contraste con el estudio actual, Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. informaron que, en el grupo alimentado con lupino de hojas estrechas, la altura de la clara espesa, el valor de Haugh y el color de la yema fueron mayores que en el grupo control. Tales diferencias podrían deberse a la variedad de lupino utilizada por los distintos investigadores.

De acuerdo con los hallazgos actuales, Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. y Kponouglo et al. (2024)Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. informaron que las dietas que contenían semillas de lupino de hoja estrecha y fermentadas no tenían efecto en la altura de la clara y los valores de la unidad Haugh. En otro estudio, Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. informaron mayores valores de la unidad Haugh que los observados en el presente estudio para gallinas ponedoras marrones alimentadas con diferentes niveles de semillas de lupino sin procesar (Lupinus angustifolius L.). Aunque no significativo, el color de la yema aumentó linealmente con el incremento de las dietas de tratamiento, lo cual concuerda con los informes de Park et al. (2016)Park, J.H., Lee, S.I. & Kim, I.H. (2016). Effects of lupin seed supplementation on egg production performance, and qualitative egg traits in laying hens. Veterinární Medicína, 61(12): 701-709, ISSN: 1805-9392. https://doi.org/10.17221/330/2014-VETMED. , quienes reportaron que la suplementación con semillas de lupino sin procesar en dietas de gallinas ponedoras mejoraba linealmente el color de la yema. Rutkowski et al. (2017)Rutkowski, A., Hejdysz, M., Kaczmarek, S., Adamski, M., Nowaczewski, S. & Jamroz, D. (2017). The effect of addition of yellow lupin seeds (Lupinus luteus L.) to laying hen diets on performance and egg quality parameters. Journal of Animal and Feed Sciences, 26(3): 247-256, ISSN: 2719-8448. https://doi.org/10.22358/jafs/76322/2017. también encontraron una mejora significativa del color de la yema en el grupo alimentado con una dieta que contenía 25 % de semillas de lupino amarillo (Lupinus luteus L.).

Se ha informado que la inclusión de harina de semillas de lupino azul en las dietas de gallinas ponedoras mejora los perfiles de grasa de la yema, lo cual es esencial para la salud de los consumidores (Kowalska et al. 2020Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. ). Timová et al. (2020)Timová, I., Straková, E., Všetičková, L. & Suchý, P. (2020). Impact of feeding mixture containing lupin meal on improvement of polyunsaturated fatty acids in egg yolk. Czech Journal of Animal Science, 65(08): 311-321, ISSN: 1805-9309. https://doi.org/10.17221/87/2020-CJAS. demostraron que reemplazar la soya extraída por harina de semillas de lupino blanco en las mezclas de alimento, tanto al 50 % como al 100 % como fuente de proteína, disminuyó significativamente los ácidos grasos saturados y aumentó los ácidos grasos monoinsaturados y poliinsaturados, los cuales son esenciales para la salud de los consumidores, especialmente para la prevención de enfermedades coronarias. Los niveles de colesterol de los huevos producidos por gallinas alimentadas con semillas fermentadas de lupino (Lupinus angustifolius) fueron significativamente más bajos que los de la dieta de control (Kponouglo et al. 2024Kponouglo, K., Koné, G.A., Good, M., Grosset, N., Gautier, M. & Kouba, M. (2024). Effect of using germinated and fermented lupin and oats as a dietary protein source on laying hen performance and egg quality. Agriculture, 14(11): 1942, ISSN: 2077-0472. https://doi.org/10.3390/agriculture14111942. ). De manera similar, Lim y Choi (2023)Lim, C.I. & Choi, N.J. (2023). Effect of lupin (Lupinus angustifolius) as a soybean meal replacement on the performance, meat quality, and blood parameters of broilers. Canadian Journal of Animal Science, 103(2): 167-173, ISSN: 1918-1825. https://dx.doi.org/10.1139/CJAS-2022-0104. observaron niveles significativamente mayores de ácidos grasos poliinsaturados en pollos de ceba alimentados con 10 % y 20 % de harina de semillas de lupino (Lupinus angustifolius), en comparación con aquellos alimentados con la dieta control.

Efecto de la edad en la calidad del huevo

Como se muestra en la tabla 6, el peso de la cáscara seca y el color de la yema no se afectaron por la edad, mientras que el peso del huevo, la longitud del huevo, el ancho del huevo, la altura y el ancho de la yema, la altura del albumen y el valor de la unidad Haugh aumentaron de manera significativa y lineal con la edad. Estas observaciones coinciden con las reportadas por Yurtseven et al. (2021)Yurtseven, E.P., Şekeroğlu, A., Tainika, B., Duman, M. & Şentürk, Y.E. (2021). Effect of production system and age on egg quality parameters: A case of Niğde Province Çamardı District, Turkey. Turkish Journal of Agriculture-Food Science and Technology, 9(8): 1407-1412, ISSN: 2148-127X. https://doi.org/10.24925/turjaf.v9i8.1407-1412.4241. en gallinas criadas en el sistema de producción de campo abierto. De manera similar, Manyeula et al. (2021)Manyeula, F., Sebolai, B., Sempule, G. & Moreki, J.C. (2021). Effects of broiler breeders’ Age on egg quality characteristics and their correlation coefficients. Journal of World's Poultry Research, 11(3): 368-375, ISSN: 2322-455X. https://dx.doi.org/10.36380/jwpr.2021.44. reportaron que el peso del huevo, la longitud del huevo, el ancho del huevo, la yema, el contenido del huevo, el volumen del huevo, el porcentaje de cáscara, el peso del albumen y el área superficial del huevo aumentaron con la edad de la gallina. Por otro lado, los índices de forma del huevo y de la yema disminuyeron significativamente con la edad avanzada de las gallinas y estas observaciones coinciden con las reportadas por Kraus et al. (2021)Kraus, A., Zita, L., Krunt, O., Härtlová, H. & Chmelíková, E. (2021). Determination of selected biochemical parameters in blood serum and egg quality of Czech and Slovak native hens depending on the housing system and hen age. Poultry Science, 100(2): 1142-1153, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2020.10.039. y Tainika et al. (2024)Tainika, B., Şekeroğlu, A., Akyol, A., Şentürk, Y.E., Abaci, S.H. & Duman, M. (2024). Effects of age, housing environment, and strain on physical egg quality parameters of laying hens. Brazilian Journal of Poultry Science, 26(03): 001-014, ISSN: 1806-9061. eRBCA-2024. http://dx.doi.org/10.1590/1806-9061-2024-1911. . Estos últimos autores informaron una disminución significativa de la resistencia de la cáscara con la edad de las gallinas, mientras que el grosor de la cáscara mostró un patrón inconsistente. En otro estudio, Lee et al. (2016b)Lee, M.H., Cho, E.J., Choi, E.S., Bang, M.H. & Sohn, S.H. (2016b). The effect of hen age on egg quality in commercial layer. Korean Journal of Poultry Science, 43(4): 253-261, ISSN: 2287-5387. http://dx.doi.org/10.5536/KJPS.2016.43.4.253. , Kowalska et al. (2020)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J. & Adamski, M. (2020). Quality of eggs, concentration of lysozyme in albumen, and fatty acids in yolk in relation to blue lupin-rich diet and production cycle. Animals, 10(4): 735, ISSN: 2076-2615. https://doi.org/10.3390/ani10040735. y Sam (2023)Sam, I.M. (2023). The influence of Isa Brown laying hen age on egg quality in humid tropics. Animal Research International, 20(2): 4966-4972, ISSN: 1597-3115. https://oaji.net. reportaron una disminución significativa en la altura del albumen, el valor de la unidad Haugh y el color de la yema con el aumento de la edad de las gallinas.

Manyeula et al. (2021)Manyeula, F., Sebolai, B., Sempule, G. & Moreki, J.C. (2021). Effects of broiler breeders’ Age on egg quality characteristics and their correlation coefficients. Journal of World's Poultry Research, 11(3): 368-375, ISSN: 2322-455X. https://dx.doi.org/10.36380/jwpr.2021.44. informaron aumento del índice de forma del huevo con la edad de la gallina, lo cual contrasta con el presente estudio. Tales variaciones podrían deberse a la raza de pollo y al tipo de dieta utilizada, así como a la edad de las gallinas cuando se recopilaron los datos. Por ejemplo, Lee et al. (2016b)Lee, M.H., Cho, E.J., Choi, E.S., Bang, M.H. & Sohn, S.H. (2016b). The effect of hen age on egg quality in commercial layer. Korean Journal of Poultry Science, 43(4): 253-261, ISSN: 2287-5387. http://dx.doi.org/10.5536/KJPS.2016.43.4.253. y Alo et al. (2024)Alo, E.T., Daramola, J.O., Wheto M. & Oke, O.E. (2024). Impact of broiler breeder hens’ age and egg storage on egg quality, embryonic development, and hatching traits of FUNAAB-alpha chickens. Poultry Science, 103: 103313, ISSN: 1525-3171. https://doi.org/10.1016/j.psj.2023.103313. encontraron que los huevos de gallinas de 60 semanas de edad tenían valores de altura de albumen y de unidad Haugh más bajos en comparación con los huevos de gallinas de 30 semanas. Chung y Lee (2014)Chung, S.H. & Lee, K.W. (2014). Effect of hen age, storage duration and temperature on egg quality in laying hens. International Journal of Poultry Science, 13: 634-636, ISSN: 1994-7992. https://doi.org/10.3923/ijps.2014.634.636. informaron que la edad de la gallina no afectó la calidad del huevo. Por el contrario, Kowalska et al. (2021)Kowalska, E., Kucharska-Gaca, J., Kuźniacka, J., Lewko, L., Gornowicz, E., Biesek, J., & Adamski, M. (2021). Egg quality depending on the diet with different sources of protein and age of the hens. Scientific Reports, 11(1): 2638, ISSN: 2045-2322. https://doi.org/10.1038/s41598-021-82313-1. encontraron una diferencia significativa en las características de calidad del huevo según la edad de las gallinas. El peso del huevo, la densidad de la cáscara, el peso de la cáscara, la resistencia a la rotura y el grosor aumentaron significativamente con la edad de las gallinas (de 19 a 39 semanas de edad). Los mismos autores informaron una disminución significativa en la altura del albumen y en el valor de la unidad Haugh, lo cual no concuerda con los resultados de la investigación actual.

El valor de la unidad Haugh se ha aceptado ampliamente como el “estándar de oro” para cuantificar la calidad interna y la frescura de los huevos. El valor de la unidad Haugh disminuye rápidamente con el tiempo, lo que la convierte en una medida sensible del deterioro del huevo poco después de la puesta. Por otro lado, el índice de la yema cuantifica la integridad estructural de la yema del huevo, la cual disminuye gradualmente durante el período de almacenamiento, lo que permite identificar diferencias de calidad entre huevos que se han deteriorado con el tiempo (Yurtseven et al. 2021Yurtseven, E.P., Şekeroğlu, A., Tainika, B., Duman, M. & Şentürk, Y.E. (2021). Effect of production system and age on egg quality parameters: A case of Niğde Province Çamardı District, Turkey. Turkish Journal of Agriculture-Food Science and Technology, 9(8): 1407-1412, ISSN: 2148-127X. https://doi.org/10.24925/turjaf.v9i8.1407-1412.4241. ). En contraste, se encontró que el efecto de la edad en el color de la yema en el presente estudio fue insignificante. El color de la yema se afectó debido la dieta de las aves, principalmente por la presencia de xantofilas, lo que resulta en variaciones en la intensidad del color de la yema (Lordelo et al. 2017Lordelo, M., Fernandes, E., Bessa, R.J.B. & Alves, S.P. (2017). Quality of eggs from different laying hen production systems, from indigenous breeds and specialty eggs. Poultry Science, 96(5): 1485-1491, ISSN: 1525-3171. http://dx.doi.org/10.3382/ps/pew409. ).