Evaluation of growth, sex (male proportion; sexual dimorphism), and color segregation in four cross combinations of different strains of XX female and YY male Nile Tilapia

Correspondence Noel D. Novelo, Aquaculture Research Center, Kentucky State University, Frankfort, KY 40601, USA. Email: noel.novelo@kysu.edu Abstract Four cross combinations of different YY male and female Nile Tilapia Oreochromis niloticus strains were evaluated for growth, sex, and color segregation. Red color parental strains included blotched phenotypes. The Genetically Improved Farmed Tilapia (GIFT) was the only dark (wildtype) color parental strain. Fish of the same age and cross were stocked in three replicate tanks for four crosses in one recirculating system for 167 days. Data recorded included feed consumed, body weight, total length, color, sex, and fillet weight. YY males crossed with GIFT females (Cross 2) exhibited superior growth that was significantly different (p < .05) to other three crosses. Male proportions were 79–100%. Only YY males crossed with the LSA female strain (Cross 4) yielded 100% males, but, Cross 4's productivity was inferior to that of Cross 2. Body weight advantage of males over females was 28.7–84.2%. Color segregation indicated that red color trait in Nile Tilapia is autosomal dominant, and black patch coverage was variable. This study showed that different parental strain combinations clearly impact productivity traits, and that YY male technology combined with crossbreeding provide the opportunity for genetic improvement and development of commercially beneficial superior traits in Nile Tilapia. Received: 19 February 2020 Revised: 2 July 2020 Accepted: 13 August 2020


| Study site, broodstock, spawning, and nursing
This research was conducted in clear-water, indoor, recirculating aquaculture systems (RAS) at the Aquaculture Research Center of Kentucky State University in Frankfort, KY. The strain designation, color (as described by vendor), vendor, and other characteristic details of the broodstock used in four cross combinations are listed in Table 1.
The color phenotype of red broodstock strains in this study included fish with red body color without black spots or patches, and red body color with a small patch of black pigmentation (Table 1). Parental strains used for spawning were stocked at a ratio of 3-5 females to one male in four recirculating systems on March 6, 2017 (Table 1). Each spawning system included a 1893-L, flat-bottomed, circular tank (Polytank, Inc., Litchfield, MN); a biofilter, Model T400 (Waterco USA, Augusta, GA); a submersible pump, Danner Model 9.5B (Amazon.com LLC, Seattle, WA); a 1,000 W submersible heater and thermostat (Pentair Aquatic Eco-Systems, Inc, Apopka, FL); and, one 3000-Lumen LED work light (Utilitech, Romulus, MI). The water volume of each spawning tank was kept at 1184-L. A photoperiod of 12 h light/12 h dark and a water temperature of 28 C were maintained during spawning.
Broodfish were fed 4.8-mm tilapia feed (Triton 3606, Cargill Animal Nutrition, Albany, NY) at 0.5-1% body weight/day during spawning. Females in each spawning system were checked every 2 weeks for the presence of eggs in the oral cavity, but it was not until 8 weeks after initial stocking that eggs were collected from all four spawning systems on the same day (May 8, 2017). The eggs obtained from at least two fish from each of the four crosses were combined, and eggs from each of the four crosses were incubated separately in McDonald-type hatching jars (Pentair Aquatic Eco-Systems, Inc. Apopka, FL) in a recirculating trough system kept at 28 C. Swim-up fry were fed 0.3 to 0.4-mm feed (Aquaxcel 5014; Cargill Animal Nutrition, Albany, NY) four to six times/day up to 30 days post hatch (dph). Fry from each cross (185-195 fry/cross) were then moved to four 416-L tanks and kept separate in another recirculating system. They were fed 0.6-to 0.8-mm and 1.5-mm feed (Aquaxcel 5014 and Aquaxcel 4512; Cargill Animal Nutrition, Albany, NY) four times/day until they were 71 dph.
T A B L E 1 Strain designation, color (as defined by vendor), vendor, sex, number of fish, and the mean (±SD) of the body weight (BW, g) and total length (TL, cm) of five Nile Tilapia Oreochromis niloticus parental strains used in four cross combinations . Fish were fed to apparent satiation for each feeding session. The minimum time between consecutive feeding sessions during the day was 4 hr to allow for gastric evacuation and appetite return (Riche, Haley, Oetker, Garbrecht, & Garling, 2004).
Water quality parameters measured during the comparative raising period were: temperature, dissolved oxygen, pH, and salinity (two to three times a week) using a ProDSS Multiparameter Meter (YSI Incorporated, Yellow Springs, OH); and total ammonia nitrogen, nitrite, and alkalinity (one to two times a week) using the DR3900 Spectrophotometer (Hach Company, Loveland, CO).

| Data collection
Feed was weighed in aliquots of 35, 30, 20, 15, 10, and 5 g in multiple-labeled weigh boats prior to each feeding session. Feed was given one aliquot at a time to each tank. As soon as one aliquot of feed was eaten (0-20 pellets remaining), another aliquot of equal or smaller weight was supplied. Fish actively ate larger aliquots (35, 30, and 20 g) more frequently in the first 30-40 min of feeding, and smaller feed portions (15, 10, or 5 g) were given thereafter until feeding activity ceased.
The body weight (g) and total length (cm) were measured from a random sample of fish for each tank on July were processed to obtain the skinless fillet weight (g).
The color phenotype was recorded as either dark (wild-type), solid red (no black pigmentation on body surface), or as red-black (RB) for fish with red body color with presence of black pigmentation on the body surface (blotched phenotype). Fish exhibiting the blotched phenotype were assigned to one of three qualitative categories to characterize variability of black pigmentation: RB1 = red body color with presence of one small patch of black pigmentation; RB2 = red body color with presence of two or three small patches of black pigmentation in different areas; and RB3 = red body color with two or more large black patches that were distributed over large areas.

| Growth parameter calculations
Survival rate in each tank was calculated as a percentage for the number of fish collected during harvest from the number of fish stocked. The mean body weight in each tank was calculated for the day of stocking and for five subsequent sampling dates. Weight gain for 167 days in each tank was calculated as WG = W f − W i , where W f = the final mean body weight and W i = initial mean body weight. Daily growth rate (g/day) in each tank was calculated as W G /t, where W G = weight gain (g) at 167 days, and t = 167 days. The number of feed aliquots and their weights were recorded for each tank for each feeding session, and total feed consumed was calculated as the sum of aliquot weights fed to each tank for each feeding session during 167 days. The Feed Conversion Ratio (FCR) for the 167 days in each tank was calculated as F/WG, where F = total feed fed and WG = weight gain at 167 days. The body condition factor 'K' was calculated as (W/TL 3 ) × 100, where W = body weight (g) and TL = the total length (cm) of each fish at harvest. Fillet yield (%) was calculated as fillet weight/total body weight × 100. The number of males and females for each cross were recorded for investigation of sex segregation, and their body weight (g) at harvest was used for evaluation of sexual dimorphism.
The body weight advantage of males over females in crosses was calculated (as a percentage) as the difference between mean weight of males and females divided by the mean weight of females multiplied by 100.

| Statistical analysis
The SAS ® University Edition software (SAS Institute Inc., Cary, NC) was used for statistical analysis. The dependent variables of daily growth rate (g/d), feed consumed, FCR, body condition factor, and fillet yield after 167 days of raising were analyzed as a response to the treatment of parental strain combination (hereafter cross) as either a linear model or a nonparametric one-way test, depending on the whether the residuals of the linear model were normally 3 | RESULTS

| Comparative raising and growth
Water quality measurements (mean ± SD) during the comparative raising period were: 27 ± 1 C water temperature; 6.0 ± 0.8 mg/L dissolved oxygen; 7.98 ± 0.29 pH; 0.23 ± 0.30 mg/L total ammonia nitrogen; 0.19 ± 0.15 mg/L nitrite; 120 ± 30 mg/L alkalinity; and, 2.4 ± 0.7 ppt salinity. Survival ranged from 99 to 100% (Table 2). Cross, sampling day, and the interaction of cross and sampling day had a significant (p < .0001) effect on body weight ( Figure 1). Cross 2 (Til-Aqua YY males × GIFT females) had the highest mean body weight, and this was significantly different (p < .0001) to that of Cross 1 (Til-Aqua YY males × Miami females), Cross 3 (Til-Aqua YY males × LSA females), and Cross 4 (Fishgen YY males × LSA females) throughout 167 days of growth (Table 2 and Figure 1). Cross 2 grew 75% larger and 1.8 times faster than Cross 1, 62% larger and 1.7 times faster than Cross 3, and 57% larger and 1.6 times faster than Cross 4 as measured by final body weight and daily growth rate (Table 2). Cross 2 had the highest mean daily growth rate (4.08 g/d); this was significantly different (p < .0001) from that of the other crosses (2.28 g/d, Cross 1; 2.35 g/d, Cross 3; and 2.57 g/d, Cross 4) ( Table 2).

| Male proportion and sexual dimorphism
Data on sex segregation and harvest weight by sex are presented in Table 3. Only Cross 4 (Fishgen YY males × LSA females) comprised 100% males; and, Crosses 1, 2, and 3 (Til-Aqua YY males crossed with females from three distinct strains) were 79-85% males (Table 3). Although females were identified in these three crosses, no evidence of reproduction (presence of eggs or fry) was observed at any time. The effect of cross, sex, and the interaction of cross and sex on body weight at harvest were significant (p < .05). The mean body weight of males was larger and significantly different (p < .0001) compared to their female cohorts in Crosses 1, 2, and 3; and, the body weight advantage of males ranged from 28.7 to 84.2% (Table 3). Although females were significantly smaller than their male cohorts, T A B L E 2 Growth parameters (mean ± SD) of Nile Tilapia crosses measured for 167 days of comparative raising were: (i) initial body weight (BW I , g), (ii) final body weight (BW F , g), (iii) survival (%), (iv) daily growth rate (g/d), (v) feed consumed (kg), (vi) feed conversion ratio (FCR), (vii) body condition factor (K), and (viii) percent fillet yield (FY, %). Significant differences (p < .05) among crosses were indicated by different lowercase letters 'x, y, and z'  F I G U R E 1 Growth of Nile Tilapia crosses during 167 d period. Significant differences (p < .05) in body weight among the crosses for six sampling days were indicated by different letter superscripts Cross 2 female mean body weight (472 g) was superior to that of males (422 g) and females (328 g) in Cross 1 and to females in Cross 3 (253 g) (Table 3); and, Cross 2 female mean body weight was similar to that of males in Cross 3 (466 g) and Cross 4 (447 g) (Table 3).

| Color segregation
Data on color segregation of the four parental strain combinations were presented in Table 4. No dark (wild-type) body color phenotype was observed in any fish in any of the four crosses. Two distinct red body color phenotypes were observed: (a) solid red (complete absence of black pigmentation on body surface); and (b) blotched (red body color with variable presence of black pigmentation) ( Table 4). The solid red body color phenotype comprised 48.3% of Cross 1, 44.0% of Cross 3, and 10.0% of Cross 4. The remainder of Cross 1 and Cross 3 mostly exhibited the "RB1" blotched phenotype (a red body color with the presence of one small patch of black pigmentation), while in Cross 4 a higher number of fish exhibited the "RB2" blotched phenotype (red body color with presence of two or three small patches of black pigmentation) ( Table 4). The blotched phenotype comprised 100% of Cross 2, with 96.6% of fish exhibiting the highest degree of dark pigmented coverage represented by the "RB3" phenotype (red body color with two or more large black patches distributed over large areas) (Table 4).
T A B L E 3 Sex segregation (%) and sexual dimorphism in body weight (mean ± SD) of Nile Tilapia crosses. Significant differences (p < .05) in body weight were indicated by the letters "a and b" for males and female from the same cross  Ridha, 2006aRidha, , 2006b. Multiple growth parameters evaluated strongly indicated that Til-Aqua YY males crossed with females from the Genetically Improved Farmed Tilapia (GIFT) strain (Cross 2) exhibited superior productivity traits compared to the other three crosses tested.
Cross 2 yielded a higher mean daily growth rate (4.08 g/day for 167 days; 50 fish/0.95 m 3 ) than that previously reported for different Nile Tilapia strains (Arredondo-Figueroa et al., 2015, Ridha, 2006a, 2006b 120 fish/m 3 ), 5.06 g/day (60 days; 80 fish/m 3 ), and 7.23 g/day (60 days; 60 fish/m 3 ); and, the mean daily growth rate was 4.9 g/day for 180 days (dos Santos et al., 2019). In comparison to previous studies, Cross 2 exhibited a superior growth rate advantage not often reported, and the other three crosses exhibited growth rates that were similar to those previously reported for Nile Tilapia reared in RAS.
In terms of feed conversion efficiency, fish can achieve higher growth rates by (i) decreasing the amount of food consumed in relation to weight gain; (ii) increasing the quantity of feed consumed as a result of increased appetite; or (iii) by a combination of effective feed utilization and higher feed consumption (Gomelsky, 2011 Santos et al., 2019;Ridha, 2006aRidha, , 2006b. Although the body condition factor has not frequently been reported in Nile Tilapia RAS-based growth studies, the values reported in this study (2.14-2.49) were higher than values in a nutrition study (1.83-2.01) on Nile Tilapia (Herath, Haga, & Satoh, 2016). The fillet yield obtained in Cross 2 (36%) was within the higher end of the range

| Male proportion and sexual dimorphism
Sexual size dimorphism in this study varied widely as shown in body weight advantage (29-84%) of males to females. The range of variation in sexual size dimorphism was similar to that previously reported for seven tilapia strains (Lind et al., 2015). Data on sexual dimorphism and growth parameters obtained in the present study showed that 100% male proportion (Cross 4) did not inherently result in optimal production, and that the effect of the parental strain combination used to produce Cross 2 was significant in yielding fast-growing and predominantly male tilapia.

McAndrew et al. (1988) have performed a comprehensive investigation of inheritance and expression of red color in
Nile tilapia. The results of that study showed that the red body color in this species is controlled by a dominant allele R of one gene (R/r). Fish homozygous for recessive allele (genotype rr) have dark (wild-type) color type while fish with genotypes RR and Rr can have either solid red (without black spots or patches) or red-black (blotched) body color.
Blotching is a very variable trait; the black patches can cover up to about 25% of the fish surface. The degree of blotching is reduced with increase of the number dominant allele R in fish genotype. Fish with genotype RR have lower degree of blotching than heterozygotes Rr; however, the ranges of variability of this trait between RR and Rr fish are overlapping (McAndrew et al., 1988). In further studies, Mather, Lal, and Wilson (2001), Garduño-Lugo, Muñoz-Córdova, and Olvera-Novoa (2004) and Thodesen et al. (2013) reported a decrease in intensity of black blotching in red tilapia by selection applied in several consecutive generations while Hilsdorf, Penman, Farias, and McAndrew (2002), Rajaee (2011) and Lago et al. (2019) have described development and quantification of black pigmentation in red tilapia.
As mentioned above, the Til-Aqua YY, Fishgen YY and Miami parental strains were marketed and acquired as 'red' fish for the present study. In reality, fish from these strains were either red (R category-fish with red body color with no black pigmentation) or had minor expression of black patches (RB1 category) ( Table 4). The presence of black spots on an otherwise basic red phenotype is a common characteristic of red tilapia stocks (Mather et al., 2001). No dark (wild type) fish were recorded in Cross 2, which was obtained by crossing Til-Aqua males (R and RB1 categories) with dark (wild type) GIFT females. This shows that in Nile tilapia used in the present study, the same as in experiments by McAndrew et al. (1988), the red color is controlled by a dominant mutation. The absence of dark fish in Cross 2 showed also that parental Til-Aqua males were homozygous for dominant allele (genotype RR) but not heterozygous (Rr) (see Table 4). No solid red (R category) and blotched fish with minor development of black patches (RB1 category) were present in Cross 2 while 96.6% of blotched fish in this cross belonged to RB3 category with highest level of black patches development. This is in agreement with an observation by McAndrew et al. (1988) that fish, which are originated from crosses of wild-type fish, have most intense blotch patterns. As mentioned above, the LSA parental strain was originated from crossing of red fish with wild-type (dark) fish and was heavily blotched. This indicates that LSA females used for production of Crosses 3 and 4 were obviously heterozygous for red color gene (genotype Rr). Because no dark (wild type) fish were observed in Crosses 3 and 4 it can be suggested that parental YY males for these crosses (Til-Aqua and Fishgen YY males, respectively) were homozygous for dominant allele (genotype RR) but not heterozygous (genotype Rr) (see Table 4). In Crosses 1, 3, and 4 the offspring could have genotypes RR and Rr (Table 4); however, it is impossible to determine exactly the genotype of every fish in crosses because, as was shown by McAndrew et al. (1988), the rate of blotching in fish with these genotypes overlaps. Seg-

| Genetic differentiation of parental strains
Recently, Delomas, Gomelsky, Vu, Campbell, and Novelo (2019) reported data on genetic differentiation of eight Nile Tilapia strains including those that were used as broodstock in this study. Nile Tilapia strains were analyzed based on biallelic single-nucleotide polymorphisms (SNPs), and genetic differentiation was measured by pairwise fixation index (F ST ) in which values increase with the increase in genetic distance between strains (Delomas et al., 2019). The highest value of pairwise F ST (0.24) was observed between parental strains used for production of Cross 1 (Til-Aqua YY males x Miami females), the intermediate value of F ST (0.15) was detected between strains used for production of Cross 2 (Til-Aqua YY males × GIFT females), and the smallest level of genetic differentiation (F ST = 0.09) was observed between parental strains used for production of Cross 3 (Til-Aqua YY males × LSA females) and Cross 4 (Fishgen YY males × LSA females) (Delomas et al., 2019). The results of the present study showed that Cross 2 had highest productivity, Cross 1 was the least productive, and Crosses 3 and 4 demonstrated intermediate productivity.
The data obtained in these two studies indicate that performance of the tested inter-strain crosses may not depend on the level of genetic differentiation between parental strains. The absence of dependence between pairwise F ST values for the strains and the performance data obtained in the present study contradicts the suggestion that the rate of heterosis increases with increase of genetic distance between strains used in crosses (Shikano & Taniguchi, 2002). However, the results of the present study support the proposition that the effect of heterosis vary between different strains and the possibility of heterosis needs to be determined on a case-by-case basis (Gjedrem & Robinson, 2014).

| Practical implications and recommendations
This study was designed to test the combination of YY male technology and crossbreeding for identifying potential gains in growth and for investigating sex and color. The results provided strong evidence that the genetic contribution of different parental strain combinations clearly impact commercially important productivity traits, and that crossing YY males with females from genetically improved tilapia strains such as the GIFT strain provide the opportunity to produce predominantly male, fast-growing fish of superior size. This shows the importance of the genetic material (species and strain) used to obtain highly beneficial crosses for commercial use; thus, it is strongly advisable that in order to improve tilapia production and profitability, suppliers, and farmers should ascertain the genetic background and productivity of fish they supply or purchase for hatchery (seedstock) and grow-out purposes.
The ability to raise tilapia such as those obtained from Cross 2 is of high potential benefit for different commercial production systems such as in RAS for maximum control of culture parameters, and such as in cage culture in ponds to maximize use of pond resources in temperate climate regions such as Kentucky that have a limited time window for growth (110-120 days) during summer (Danaher, Tidwell, Coyle, Dasgupta, & Zimba, 2007). It is important to note that though no reproduction was observed during rearing of these four crosses in this study, reproduction is possible in unconfined stocking in ponds; thus, tilapia such as Cross 2 that may be predominantly but not 100% male should be raised in culture environments (such as in cages in ponds) that inhibit reproduction (Bentsen et al., 2012;Danaher et al., 2007;Lind et al., 2015;Tidwell, Coyle, & Bright, 2010).
Studies on red color tilapia report the high cultural, aesthetic, and commercial value of red tilapia (Ng & Hanim, 2007;Pongthana et al., 2010;Ramírez-Paredes et al., 2012), and the negative effect on appearance of the blotched phenotype (black patches) on red body color of tilapia that, by implication, has negative market appeal (Garduño-Lugo et al., 2004;Mather et al., 2001;McAndrew et al., 1988;Thodesen et al., 2013). Based on these suggestions on red color tilapia marketing trends, solid red fish and fish with minimal expression of blotching (e.g., RB1 and RB2) such as in Crosses 1, 3, and 4 may be more desirable for marketing as live or dress-out (gilled, gutted and scaled) products, while Cross 2 may be considered more appropriate as a fillet product. However, marketing and sales data on tilapia is insufficient (and not readily available, or reported), and researchers, farmers, and consumers would benefit from studies investigating acceptance and willingness to pay for red color morphs (complete red coverage; and, red color fish with RB1, RB2, and RB3 type blotched phenotypes), and wild-type color (dark) fish in whole fish markets. In addition, future studies should continue efforts at genetic improvement of tilapia by combining practical genetic approaches such as YY male technology and crossbreeding, and by investigating the productivity of crosses between dark (wild-type) color YY males and dark color females from genetically improved fish such as the GIFT strain.

ACKNOWLEDGMENTS
This study was supported by the USDA/NIFA Grant 2015-38821-24389 to Kentucky State University. We thank Dr. Thomas Delomas for valuable suggestions and Dr. Michael D. Kaller for help in statistical analysis.