Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)

19/August/2019 Approved: 23/November/2019 ABSTRACT This investigation was carried out to determine the effect of Essential Fatty Acids proportion (EFAs [n-6, n-3]) in feed through the mixture of soy, olive, canola or chia oil on EFA profile in eggs as well as productive and reproductive performance of Japanese quail. We used 120 quail from 7 to 22 weeks of age, in 15 cages in groups of 6 females and 2 males assigned according to the completely randomized design to 3 treatments with 5 replicates. The treatments were n-6:n-3 proportions 10:1 (control), 4:1 and 1:1. FA profile in yolk, feed intake, laying rate, egg weight, fertility, hatchability, and embryonic mortality were measured. In the egg yolk, n-6 content was similar in the proportions ( p> 0.05), while n-3 content increased ( p< 0.01) as n-6:n-3 ratio decreased in the feed. Feed consumption per quail was similar between treatments ( p> 0.05). In 4:1 and 1:1 proportion laying percentage was greater, but egg weight was lower ( p< 0.01). Fertility and hatchability were similar between proportions n-6, n-3 ( p> 0.68). Early and total embryonic mortality was lower in 10:1 and 4:1 proportion ( p< 0.01); while intermediate and late mortality was similar ( p> 0.30). The results of the experiment indicate that


INTRODUCTION
During the incubation process in birds, egg yolk lipids are the energy reserves and provide the embryo the essential fatty acids (Cherian, 2015), necessary for the formation of cell membranes (Cherian et al., 1997). The polyunsaturated fatty acids (PUFA) linoleic acid (AL 18: 2n-6) and α-linolenic acid (ALA; 18: 3n-3) are obtained by birds in feed. However, the ability to incorporate n-3 to the yolk can vary according to the source of PUFA and bird type: chicken, quail, turkey or geese (Nadia et al., 2012). Chickens have the liver enzymes delta-6-desaturase and delta-5-desaturase that allows them to synthesize from linolenic acid (n-3), eicosapentaenoic acid and docosahexaenoic acid (DHA) (Barceló-Coblijn & Murphy, 2009), and from linoleic acid (n-6) arachidonic acid (AA) (Spector, 2000); however, n-6 and n-3 compete for liver enzymes in the biochemical pathways of desaturation and elongation (Jing et al., 2013). AA and DHA are important during the post-hatching period due to rapid cell proliferation and intense tissue accumulation of these during this time (Cherian & Sim, 1992), as well as favoring the maturation of lymphoid organs (Cherian et al., 1997), therefore their function is likely important during incubation (Cherian & Sim, 1992).
The Japanese quail (Coturnix coturnix japonica) is native to Europe, North Africa and Asia, (Neumann, 2001); it is of rapid growth, precocity,

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014 resistance to diseases and high productivity (Lucotte, 1980). Quail meat production is concentrated in Spain, France, and the United States, and egg production in China, Japan and Brazil (Minvielle, 2004). Studies have been conducted in quails to enrich the egg with n-3 by including up to 4% fish oil or flaxseed in feed (Güçlü, 2008;Al-Daraji et al., 2010). However, the results of supplementation with sources containing n-3 showed no effect on fertility and hatchability of lightweight breeder hens (Nadia et al., 2012) or quails (Manohar, 2017); and even decreased in heavy reproductive hens (Herstad et al., 2000). There was increased fertility in turkey (Shamma et al., 2016) and quail hens (Manohar, 2017), as well as an increase in hatchability in quails (Al-Daraji et al., 2010;Manohar, 2017). Al-Daraji et al. (2010) observed that quail fertility and hatchability improved by including 3% fish or flaxseed oil compared to sunflower oil. It is possible to attribute this effect to the narrowest n-6:n-3 proportion in fish and flaxseed oil than in sunflower oil. However, in the studies conducted, the effect of the source of EFA was determined regardless of the n-6:n-3 proportion; therefore, the objective of this study was to determine the effect of n-6:n-3 proportion in the feed on EFA profile of the egg as well as productive and reproductive performance of Japanese quail.

MATERIALS AND METHODS
The experiment was carried out in the Poultry Unit of the Facultad de Medicina Veterinaria y Zootecnia of the Universidad Autonoma de Sinaloa,in Culiacan Sinaloa,Mexico (24 46'13'' LN and 107 21'14'' LO). The climate of the zone is BS (h') w (w) (e), semi-dry very hot, with rains in the summer, Köppen classification; with an annual average temperature of 25.9 ºC; average relative humidity of 68%, maximum of 81% and minimum 51%; average annual precipitation of 688.5 mm.
The experiment was conducted according to the technical specifications for the production, care and use of laboratory animals of the Mexican official standard (NOM-062-ZOO-1999); and the specifications of the Institutional Committee for the Care and Use of Animals of the Facultad de Medicina Veterinaria y Zootecnia of the Universidad Autonoma de Sinaloa (Protocol FMVZ-171/11-11-2016). The experimental period comprised four periods of 21 days for productive response and nine periods of 24 days for reproductive response. Before initiating data collection, the quails were adapted to cage management for seven days. 120 quails (90 females and 30 males) were utilized. Egg collection was performed twice a day (08:00 a.m. and 18:00 p.m.). The temperature and relative humidity (RH) in the coop was 25.2 ± 3.7°C and 40.7 ± 7.2%, respectively.
The experiment was established under a completely randomized design with three treatments corresponding to diets with n-6:n-3 proportions in feed: (control) 10:1, 4:1 and 1:1, with five replicates of 8 quails (6 females and 2 males) per treatment, and weeks as a cross-factor. The wire battery cages (60 x 50 x 20 cm) allowed 375 cm 2 per quail. The lighting period was 16 h per day and the feed and water were offered ad libitum.
To formulate the diets, fatty acid (FA) profile of soy, olive, canola or chia oil (Table 1); as well as proximal chemical composition (AOAC, 2000) of corn and soybean meal was determined. Metabolizable energy of corn and soybean meal was estimated with the equation: MS (kcal/kg) = 3.75 x crude protein + 8.09 x ether extract -6.95 x crude fiber + 3.94 x nitrogen-free extract (Moir et al., 1980). The diets (Table 2) were formulated according to the nutritional requirements for Japanese quail breeders (NRC 1994), and the composition of EFA profile of every oil was considered. A flour-based feed was prepared every week and stored in plastic boxes at 20 to 22°C, subsamples were taken from each batch of feed to determine FA (Table 3) and Proximate chemical analysis. The peroxide index in the feed (NMX-F-154-1987) was measured in the samples after 7 days of storage (Table 3).
Three weeks into the laying cycle, based on shape, size and egg color, collection and selection were initiated. The eggs to be incubated were kept at 11.0 ± 0.41°C (Nieto Refrigerator, Critotec CFX-8 Model, Guadalajara, Jalisco, Mexico). An automatic incubator (Huacuja, Model 1200, Guadalajara, Jalisco, Mexico) was used, and the eggs were maintained at 37.44 ± 0.22°C and 74.04 ± 2.08% RH for 334 hours, then they were transferred to a hatchery, where the eggs spent 3 to 4 days at 37.5 ± 0.4°C and 90.2 ± 0.41%

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014 RH. Withdrawal of chicks began around 12 h after hatching began. The unhatched eggs were broken and observed with the naked eye to determine if they were fertile, as well as the stage of embryonic death; and classified as early, intermediate, late and total embryonic death according to the classification proposed by Dalton, (2000). At week 20 of the laying cycle, three eggs from every treatment were randomly collected for FA determination.
Fatty acid profile of feed yolk and oils was carried out at the Food Technology Laboratory in the Research Center for Food Development Food in Culiacan Sinaloa utilizing the methods developed by Folch et al. (1957) and AOAC (1998) standard 963.22 with modifications; subsequently they were dry evaporated in a rotary evaporator, after methylation the filtrate was recovered in a 2 mL vial, stored in a nitrogen atmosphere and placed in the freezer. Subsequently, 1 μL of the sample was injected into a gas chromatograph. The methyl esters dissolved in hexane were analyzed with a chromatograph (Varian CP-3800, USA), with flame ionization detector (FID) equipped with Omegawax 320 column of 30 m x 0.32 mm, 0.25 mm internal diameter (Supelco , USA). Helium was used as a carrier gas at a rate of 3 mL/min. The oven temperature was maintained at 140°C for 5 minutes, presetat a maximum temperature of 240°C with an increase of 4°C every 90 seconds. Both the temperature of the injector and the detector were set at 260°C. For the identification and quantification of fatty acids, the retention time of sample was compared with those of a standard mixture consisting of 37 methyl esters of fatty acids (Supelco, Bellefonte, USA).
FA results were expressed in percentage of fatty acid with respect to the percentage of fat contained in the sample. The peroxide value was expressed in meq O 2 /kg. In productive response, after every feed consumption period, egg number and weight were recorded. For reproductive response after every egg collection, fertility rate, hatchability of fertile eggs and early, intermediate, late and total mortality were recorded.
The statistical analysis of FA results in egg yolk, feed intake, laying percentage, egg weight, fertility and hatchability were performed under a model for a completely randomized experimental design. The comparison of means was made with the Tukey test. The proportions of embryonic mortality were analyzed with the Chi-square test. The maximum alpha level to accept statistical difference was 0.05.

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014

RESULTS AND DISCUSSION
Fatty acids in the yolk FA composition in egg yolk is shown in Table 4. According to the FA group, monounsaturated FA were found to be in the highest percentage, close to 50%, due to its content of oleic and palmitoleic acids, followed by saturated FA that were present in about 30% and finally polyunsaturated FA at 20%. Saturated FA were in greater percentage (p<0.01) in the 4:1 n-6:n-3 proportion than in 10:1 and 1:1 proportions; where as myristic acid and stearic acid were detected in a similar reduced proportion between treatments; while erucic acid appeared in a greater percentage (p<0.03) in the 1:1 proportion. Monounsaturated FA content was similar (p>0.05); However, oleic and palmitoleic acids were in a greater percentage, nonetheless palmitoleic acid percentage was greater (p<0.02) in the 4:1 proportion than in the 10:1 and 1:1 proportion. Polyunsaturated FA content was similar, although the 1:1 proportion had a higher content and was close to having a statistical difference (p>0.08). Linoleic acid content had the greatest percentage and was similar between proportions (p>0.05). Linolenic and docosadienoic acids were in greater percentage (p<0.02) in the1:1 proportion, which revealed a greater percentage of n-3 fatty acids (p<0.01), in accordance to feed proportion and n-6:n-3 proportion also differed. Chen & Hsu (2003) supplemented 2 to 6% refined cod liver oil to duck hens and observed that yolk concentration of saturated fatty acids decreased and while polyunsaturated fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) increased, compared to animal fat controls. In this study the n-6:n-3 proportion in feed remained in the egg yolk. Based on the amount of n-6 and n-3 fatty acids reported by Neijat et al. (2016) in egg yolk and chicken feed after the inclusion of hemp seed or oil as a source of n-3 it can be deduced that n-6:n-3 proportions in the diets is kept constant from 1.1 to 1.5 from feed to the egg yolk; this coincides with the results of Navas et al. (2001) in bass eggs (Dicentrarchus labrax L) where there was constant of 1.2 to 1.7 from feed to the egg yolk. In addition, arachidonic acid and eicosadienoic acid were detected in the yolk and were not detected in feed analysis; this is explained by the bird's ability to lengthen fatty acid chains (Spector, 2000;Barceló-Coblijn & Murphy, 2009). It has been observed that lineages or strains can modify EFA profiles, (Mao et al., 1998). Alessandri et al. (2012) reported that slow-growing egg-type lines of chickens or layers appear to have greater efficiency in the deposition of EPA and DHA with respect to meat-type chickens since elongation is affected in part by estrogen levels. Arantes da Silva et al. (2009) after the inclusion of 5% flax seed to quail diets reported that n-3 incorporation into the yolk was 20%. Mennicken et al. (2005) made a divergent selection in chickens for n-3: n-6 proportions and mentioned that n-3 increased 34.7% in the yolk with respect to feed content. These differences in the ability of these birds to incorporate n-3 to yolk fat can vary according to n-3 source and bird species (Nadia et al., 2012), due to the competition between the enzymes involved in lengthening and desaturation of linoleic and linolenic acid. A 4:1 proportion or lower has been shown to be optimal for elongating 11 g of Table 3 -EFA composition and contribution of n-6:n-3 as well as in dexperoxide index of quail diet. Proportion n-6:n-3 Fatty acids (%) 1 Nomenclature 10:1 2 4:

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014 linolenic acid to 1 g of eicosapentaenoic acid (Nadia et al., 2012), this relationship is important in foods that have a higher linoleic acid content and lower linolenic acid content, since it will reduce the conversion to EPA which is biologically more active than linoleic acid. Therefore, the optimal intake of linoleic in relation to linolenic is crucial for normal metabolism (Simopoulos, 2000), which may be related to FA source, linseed and chia contain more LAN and algae and fish oils are a source of EPA, DHA that are not present in land-based plant or animal sources.

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014 and do not give a definite response, since Baucells et al. (2000) reported that laying rate was similar after the inclusion of fish, flaxseed, and grape oils as well as tallow, where PUFA n-6:n-3 proportions ranged from 1 to 38 in chicken feed, on the other hand, Betancourt & Díaz (2009) reported that in broader proportions (7:1) laying rate was greater than in the narrowest proportion (2:1), 93.1% and 86%, respectively. In 4:1 and 1:1 proportion egg weight was lower (p<0.01) with respect to the 10:1 proportion. These results are in agreement with those of Güclü et al. (2008) who added 4% sunflower, corn, fish, soy, sesame, olive, cotton or walnut oils to quail feed and obtained eggs with a greater weight (12 g) in the n6:n3 200:1 proportion, compared with the 53:1 and 7:1 proportions of sunflower, corn or soybean oil which weighed 11.5 and 11.3 g, respectively. The greater weight seen in the 10:1 ratio is explained by the lower laying rate (87%) since there is a genetic and phenotypic negative correlation between these two parameters; in this respect, Hagger (1994) estimated a negative genetic correlation in hens (-0.267).

Reproductive response
Results in reproductive response are shown in Table  6. In this study, fertility was similar between treatments (p>0.680). In studies where different sources containing EFA are supplemented, discrepancies on the effect on fertility are reported. Nadia et al. (2012) used 1.73% flaxseed oil in light reproductive hens, and Manohar (2017) included 4% fish oil in quails and did not find any difference. In turkeys Fertility increased by 5.39% when 2% fish oil was supplemented and by 3.43% when 2% flaxseed oil was added (Shamma et al., 2016), fertility also increased by 12.75% after the inclusion of 2% fish oil in quail diets (Manohar, 2017), however Herstad et al. (2000) with diets that had 3% recycled vegetable oil or no oil at all observed that in diets for heavy reproductive hens with n-6:n-3 proportions of 1.03:1 to 1.12:1 with 3% fish oil fertility rate decreased (76.3 to 83.7%) compared to 7.6:1 to 8.31:1 proportion (89.5 to 92.1%). The source, quantity and lipid type in the diets are important. Bleisbois et al. (1997) mentions changes in the proportions of n-6:n-3 or phospholipid ratios affect sperm membrane structure and fluidity; this can alter fertility by modifying viability and ability of the sperm to interact with the reproductive tract of the female and thereby the union of the sperm with the ovum (Bongalhardo et al., 2009).
Hatchability of fertile eggs was similar between the treatments (p>0.95). Discrepancies were also found on the effect of EFA supplementation on hatchability in the studies. Nadia et al. (2012) after the inclusion of 1.73% of flax seed oil and n-6:n-3 proportions that varied from 2:1 to 10:1 in lightweight reproductive hens; and Manohar (2017) in quails with 2% flaxseed, 4% fish and 2% and 4% linseed and fish oil combinations, did not observe differences in hatchability. Hatchability increased 3.2% and 6.17% when flaxseed or fish oil with n-6:n-3 proportions of 0.22:1 and 0.08:1 were supplemented in quails, compared to corn oil (42:1) (Al-Darji et al., 2010); Manohar (2017) supplemented quail diets with 2% fish oil and observed 5.4% greater hatchability compared to a zero oil control. On the other hand, Herstad et al. (2000) observed that in heavy reproductive hens, diets with n-6:n-3 proportions of 1.03:1 to 1.12:1 from 3% fish oil, hatchability decreased (73.2 to 77.5%) compared to 7.55:1 to 8.31:1 proportion (88.5 to 92.4%) obtained from diets with 3% of recycled vegetable oil or zero oil.
The n-6:n-3 proportion 1:1 had higher early and total embryo mortality (p<0.01), while 10:1 and 4:1 proportions were similar (p>0.05) (Figure 1). Al-Daraji et al. (2010) observed that in quails supplemented with 3% fish oil total embryonic mortality was 2.92% compared to 12.32% with 3% sunflower oil inclusion. After supplementation with fish oil n-6:n-3 proportion was narrow (0.08:1) with respect to that of sunflower oil (251:1). When EFA content is higher and more double bonds exist, greater oxidation is possible. In this study, the peroxide index in feed a week after being prepared was 4.79 mEqO 2 /kg in the n-6:n-3 1:1 Values are expressed as means ± pooled standard error, (n= 9).

Effect of Essential Fatty Acid Proportion in Feed on Productive and Reproductive Performance of Japanese Quail (Coturnix coturnix japonica)
eRBCA-2019-1014 proportion while in the 10:1 proportion a 2.01 mEqO 2 / kg (p<0.01) was recorded. Calder (2002) mentions that a greater proportion of n-3 consumption can diminish immune response due to a susceptibility to oxidation due to its unsaturation. In this study, the highest mortality in the 1:1 proportion can be attributed to greater peroxidation of linolenic acid, in addition to the possibility of peroxidation of its products. Zanini et al. (2003) observed that when the n-6:n-3 proportion in their diet was narrow because it contained 32.3% linolenic acid, fertility in cockerels decreased, however, after vitamin E was administered, fertility increased.

CONCLUSION
The results of the experiment indicate that the mixture of soy, olive, canola or chia oil, to obtain n-6:n-3 proportions of 1:1, 4:1 and 10:1 does not modify feed consumption, laying rate, egg weight, fertility or hatchability; but, 4:1 and 10:1 proportion favor a diminished embryonic mortality.
Favoring breeder bird feeds that have n-6 and n-3 proportions close to 1:1 is relative; as shown by the results of this experiment which concludes that reproduction did not improve, therefore it is recommended that n-6 and n-3 content be taken into account and estimate feed consumption in milligrams or daily ingested feed percentage, more than proportion contained in diet.