Hormonal levels of estradiol, testosterone, and progesterone at entry into lay of year 1980 vs. 2000 broiler breeder females under fast and slow release from feed restriction

Hormonal levels of estradiol, testosterone, and progesterone at entry into lay of year 1980 vs.... ABSTRACT In the mid-1960s egg production, fertility, and hatchability of broiler breeder females dropped precipitously. Due to disrupted follicle hierarchies and development of the erratic oviposition and defective eggs (EODES) syndrome. EODES was controlled by restricting feed. In the 1990s, another set of problems arose at entry of broiler breeders into lay and characterized by high mortality followed by lower peak lay and reduction in egg and chick production. These problems are induced by even slight over-feeding, and hence we termed it the “Over Feeding Complex” (OFC). We have speculated that OFC is a quasi-EODES condition, induced by the intense selection for increased breast proportion. To test this, we compared, under fast (FF) and slow (SF) release from feed restriction, body composition and reproductive performance of a broiler breeder from year 1980 (B1980) and kept without selection for performance traits since then, to a line hatched in 2000 (B2000). During the first 16 d of lay, feeding treatment had little effect on egg mass or Laying % for the B1980 birds, while for the B2000 birds, SF treatment resulted in significantly greater egg mass and Laying % compared to FF, showing that the OFC indeed manifested in this experiment. However, contrary to hypothesis, follicle hierarchies were normal for both lines under both feeding treatments. To gain further insight into the OFC syndrome, we here report levels of estradiol, testosterone, and progesterone for these line and treatment groups in the time period leading up to and into lay. A significant line × feeding treatment interaction effect was found for estradiol and testosterone, to a lesser extent for progesterone. For all 3 hormones, for B1980 levels 2 to 3 wk post entry into lay were similar and intermediate under FF and SF, but differed significantly for B2000, being much greater under SF than under FF. Thus, the hormonal effects were parallel and may explain the egg mass and Laying % effects of FF and SF in the 2 genetic types. INTRODUCTION In the mid-1960s egg production, fertility, and hatchability of broiler breeder females dropped precipitously, endangering the economic viability of the industry. The problems were shown to be due to over-production of large yellow follicles leading to disrupted follicle hierarchies and consequent development of the erratic oviposition and defective eggs (EODES) syndrome (Jaap and Muir, 1968; Van Middlekoop, 1971, 1972). It was soon found that EODES could be successfully controlled by restricting feed intake of broiler breeder females; initially during rearing, and later during the entire reproductive period (reviewed in Decuypere et al. 2006 and Renema et al. 2007). Control of EODES by restricting feed intake, led inevitably to the self-evident hypothesis that EODES was caused by ad libitum feeding of the broiler breeder hens. This was confirmed by raising broiler breeders ad libitum in suitably designed experiments (Hocking et al., 1987; Katanbaf et al., 1989a,b; Yu et al., 1992; Walzen et al., 1993). Many studies have shown that the degree of development of EODES appears to stand in monotonic relation to the degree of over-feeding relative to breeder recommendations and ad libitum (reviewed in Eitan and Soller, 2009). In the 1990s, a new set of problems arose centered at entry of the broiler breeder into lay (Robinson et al., 1995; Hocking, 1996; Meijerhof, 2005) and characterized by high mortality at onset of lay (Spradley et al., 2008) followed by lower peak lay and reduction in egg and chick production (Katanbaf et al., 1989a,b; Robinson et al., 1998; Renema et al., 2008). This set of problems is induced by even slight over-feeding at entry to lay (so called “aggressive feeding”) relative to the breeder's recommendation, and hence we have termed it the “Over Feeding Complex” (OFC, in Eitan and Soller, 2009). We have elsewhere speculated that the OFC is a quasi-EODES condition, induced by a synergistic interaction between the very severe feed restriction of the growing pullet until the start of the maturation processes at age 15 to 16 wk, and the intense selection for increased breast proportion beginning at about 1980 (Eitan and Soller, 2009). To test this hypothesis, Eitan et al. (2014) compared body composition and reproductive performance of a broiler breeder line hatched in 1980 (B1980) and kept without selection for performance traits since then, to a broiler breeder line hatched in 2000 (B2000), under fast (FF) and slow (SF) release from feed restriction. Breast proportion increased by 40%, whereas abdominal fat pad proportion decreased by 50% when comparing B1980 and B2000 birds. Interaction effects on these traits were not present. In contrast, there was a striking interaction for Laying % and egg mass for the 2 wk following onset of lay. Feeding treatment had little effect on egg mass or Laying % for the B1980 birds, while there was a very powerful positive effect of SF feeding treatment for the B2000 birds showing that the OFC indeed manifested in this experiment. However, contrary to initial hypothesis, feeding treatment did not interact with genetic type with respect to follicle development; hierarchies were normal for B1980 and B2000 birds under both feeding treatments. In order to gain further insight into the OFC syndrome, as part of the same experiment (Eitan et al., 2014), we measured and here report hormone levels for estradiol (E2), testosterone (TT), and progesterone (P4), in the time period leading up to and across entry into lay in B1980 an B2000 birds under SF and FF treatments. These are the main plasma gonadal steroids that represent the developmental status of the gonadal axis of the hen. MATERIALS AND METHODS Population In this study, 2 female broiler breeder lines were compared under fast and slow release from restriction at entry into lay. One, representing the genetic level of the year 1980; the other, representing the genetic level of the year 2000. Effects of line and feeding treatment on body composition and reproductive performance were published previously (Eitan et al., 2014), and full description of stocks, rearing procedures, and experimental feeding treatments are provided in that publication. The B1980 group consisted of 47 chicks hatched October 29, 2001, derived from an Arbor Acre broiler female line established in 1980 and maintained since then at the North Talpiot Experimental Farm of the Hebrew University of Jerusalem, without selection for broiler performance traits. The B2000 group consisted of 41 Cobb 500 chicks hatched October 25, 2001 (Brown Yavne Hatchery, Kvutzat Yavne, Israel). Experimental Feeding Treatments Food was provided ad libitum until 21 d of age for the B2000 group and 30 d for the B1980 group. Following recommendations of the “Cobb Breeder Management Guide, 1998”, food quantities were adjusted for the B2000 to reach target weights of 1580 g at 105 d and 2400 g at 147 d. Following recommendations of the “Arbor Acre Management Guide, 1980”, feed quantities for B1980 were adjusted to reach target weights of 1580 g at 105 d and 2140 g at 147 d. At 21 wk, both groups (B2000 and B1980) were divided into 2 subgroups, and each subgroup received either a FF or a SF treatment. For the FF treatment, beginning at 70 g/d for B1980 and 50 g/d for B2000, feed quantities were increased by +20 g/d for wk 22 and +10 g/d weekly thereafter, until maximum of 140 g/d. The birds were maintained at this level until all entered lay. For the SF treatment, beginning from the same amounts of feed/d as above, feed quantities were increased by +10 g/d for wk 22 and +5 g/d weekly thereafter, until maximum of 110 g/d, at which feed level all SF pullets had entered lay. Research was done before formation of university institutional animal care and use committee, but all procedures were in accord with accepted principles and guidelines for animal care (FASS, 2010). Photoperiod Chicks were brooded for 21 d in electrically heated batteries, and then transferred to floor pens until reaching suitable BW for transfer to individual cages in a light-controlled shed at 28 to 40 d, depending on line. Naturally decreasing photoperiod was provided until transfer to cages. In the shed, photoperiod was 8L:16D until 21 wk of age. At 21 wk, photoperiod was increased to 12L:12D, and by 1 h every 2 wk until 17L:7D. Sample and Data Collection The birds were divided into 4 groups of 20 to 24 birds each, according to genetic-type × feeding-treatment combination. Each group was divided into 2 replicate blocks of 10 to 12 birds each. The birds were housed in a darkout shed, in individual cages, according to the following scheme, where groups are coded A = B1980-SF, B = B1980-FF, C = B2000-SF, and D = B2000-FF; and replicates are coded 1 and 2: A1, A2: B1, B2 etc. A1-B1-C2-D2: upper row D1-C1-B2-A2: lower row Although exact time of day when samples were collected was not recorded, it was always between 9:00 to 12:00 am. Thus, any residual diurnal variation should not affect hormone levels for E4 and TT which do not show diurnal variation. However, P4 levels are known to show large diurnal variation, and this residual variation was not controlled. Thus, it may be a source of error variation for P4. That being said, the time of collection did not vary systematically among treatment groups, and consequently should not confound the results. Hormone Assays Heparinized blood samples of 2 to 3 mL were drawn from the brachial vein at 19 wk, and weekly from 21 wk. Samples were kept in ice, and taken to the Hebrew University Faculty of Agriculture at Rehovot. Plasma was stored at –20ºC until assayed. All samples were measured on the same day, and plasma E2, TT, and P4 were measured in a single assay by enzyme-linked immunosorbent assay according to a previously described method (Nash et al., 2000), which was validated for laying hens. Dilutions of primary antibody and tracer were 1:320 000 and 1:320 for E2; 1:160 000 and 1:160 for TT and 1:5000 and 1:50 for P4. For all 3 hormones, the minimal detectable dose was 0.78 pg/mL, and the inter-assay coefficients of variation were less than 5%. Statistical Analyses Since at a given chronological age every bird may be in a different physiological state relative to entering lay, changes in the mean hormone value of a population with age primarily reflect the changing proportion of birds that are in lay or close to lay, rather than the physiological state of the bird. In order to account for this, data were plotted against each individual's “physiological age”. That is, for each bird, the week of laying the first egg was taken as the origin (zero) week for measuring physiological age. Measurement dates for each bird were assigned positive (+) or negative (–) physiological age values, according to number of weeks preceding or following week of initiation of lay (zero). As compared to chronological age, the physiological age analysis has 2 weaknesses. (1) The number of samples in each “physiological week” for a given treatment group depends on the number of birds at a given removal from first egg, and thus varies much more than when analyzed according to the chronological age. Thus, the procedure introduces a degree of experimental sampling of birds in moving from week to week. (2) Photoperiod and feeding quantities were increased systematically for all birds according to chronological age. Consequently, the birds at a given physiological age can be at a different photoperiod and feeding levels, depending on the effect of their genetic type and feeding treatment on age at first egg. It should be noted, however, that after the first week, the photoperiod for all treatment groups was above the minimal duration of 10 to 13 h of light needed for entering lay (Sharp 1993). To test for statistical significance of treatment and genetic-line effects, data were analyzed as a 2-way ANOVA with genetic line, and feeding treatment as the main effects, using the Fit Model procedure of JMP version 4.01 (SAS Institute Inc., Cary, NC). To reduce fluctuations among sample groups in the ANOVA, the data of each 2 consecutive physiological ages, starting from –9 wk were combined into “Periods” of combined weeks, i.e., Period A: –8, –9 wk; Period B: –6, –7 wk; Period C: –4, –5 wk; Period D: –2, –3 wk; Period E: –1, 0, +1 wk; Period F: +2, +3 wk. ANOVA analysis for each of the 3 hormones was implemented separately for periods B, D, and F. Results for E2, TT, and P4 are presented in Table 2. When interaction effects were significant we tested subclass means for significance of differences between line × treatment groups, and examined the obtained values for the story that they tell. The usual P = 0.05 significance level was used; correction was not made for multiple tests. RESULTS Hormone Levels by Physiological Age Figures 1A, 2 and 3 show mean hormone levels by physiological age for E2, TT, and P4. Samples were taken for all birds at the same chronological start point. The treatment groups, however, entered lay at different chronological ages (as may be seen in Figure 1A for E2). Consequently, physiological start point was further from first egg for groups with later onset of sexual maturity (e.g., start point at –11 wk for B2000 SF) and closer to first egg for groups with earlier onset (e.g., –8 wk for B1980 FF and SF). Figure 1. View largeDownload slide Mean hormone levels by physiological (A) and chronological (B) age for estradiol (E2). (A) Physiological age for estradiol (E2). (B) Chronological age for estradiol (E2). Figure 1. View largeDownload slide Mean hormone levels by physiological (A) and chronological (B) age for estradiol (E2). (A) Physiological age for estradiol (E2). (B) Chronological age for estradiol (E2). Figure 2. View largeDownload slide Mean hormone levels by physiological age for testosterone. Figure 2. View largeDownload slide Mean hormone levels by physiological age for testosterone. Figure 3. View largeDownload slide Mean hormone levels by physiological age for progesterone (P4). Figure 3. View largeDownload slide Mean hormone levels by physiological age for progesterone (P4). Figure 1A shows E2 levels against physiological age. Differences among treatment groups within age groups in the weeks prior to first egg are small, indicating good technical control of measurement variation. There is a steady slow and linear rise in E2 levels from 11 wk before lay until 2 or 3 wk after onset of lay, when the birds were taken for examinations. The steady rise continues unabated across first egg and into the laying period for all except the B2000 SF group. This group shows a marked increase on entry into lay, clearly distinguishing it from the other treatment groups. It is noteworthy that after entry into lay B2000 and B1980 differ widely under SF (B2000 SF is highest group and B1980 SF is the lowest), while under FF, B2000 and B1980 are intermediate and very similar in hormone levels. Thus, feeding-treatment effect appears to depend on line, i.e., technically speaking there is an apparent feeding × line interaction effect. E2 Levels by Chronological Age Figure 1B shows E2 levels against chronological age. The much greater differences among treatment × line groups at given chronological age (Figure 1B), compared to much smaller differences among groups at given physiological age (Figure 1A), clearly justifies the use of physiological age as our parameter of interest. Figure 2 shows TT levels against physiological age. In contrast to E2, there are large differences among treatment groups at the same physiological age and across physiological age within treatment groups, particularly for B1980 FF and B2000 FF. Differences within and across physiological age groups for B2000 SF and B1980 SF were much smaller. Overall, TT levels start high and remain high until wk 0. At this point, for B1980 FF and B1980 SF, there is tendency to increase, but not dramatically. B2000 SF, however, increased markedly from being the treatment group with lowest hormone level prior to first egg, to being the treatment group with highest hormone level after first egg. The post-lay interaction pattern seen with E2 recurs here also: under SF, B2000 has highest hormone levels, B1980 the lowest, while under FF, B2000 and B1980 are similar and intermediate. Figure 3 shows mean P4 levels of the 4 treatment groups against physiological age. Hormone levels start and remain very low until –2 wk, and rise dramatically with first egg. Here too, the very tight scatter of means of the 4 groups within any given physiological age prior to first egg shows that the assay was highly reproducible and had little technical variation. Thus, the large differences among groups on entry into lay and post entry into lay can be taken to reflect the actual variation in hormone levels, for the sampled groups. This may be due in part to residual diurnal variation within the time window of sampling, and differences among the sampled birds in the same physiological-age sample groups as explained above. Here too, the same post-lay hormone level pattern can be discerned in large part: under SF, B1980 is lowest and B2000 is highest, while under FF, B1980 is intermediate. B2000 is too variable to call. Statistical Significance of Treatment × Genetic Line Effects To test for statistical significance of treatment and genetic-line effects, data were analyzed as a 2-way ANOVA, with genetic line and feeding treatment as main effects. As noted in Methods, to reduce fluctuations among sample groups in the ANOVA, the data of each 2 consecutive physiological ages, starting from –9 wk were combined into “Periods”, running from Period A: –9, –8 wk to Period F: +2, +3 wk. ANOVA analysis for each of the 3 hormones was implemented separately for periods B, D, and F. There were only a few sporadic significant main effects, none of which remained significant after Bonferroni correction for multiple tests. For Periods B and D, interaction effects were also not significant. Therefore, for these periods, data were combined across subclasses to provide overall estimates of time trends in hormone levels and were tested for longitudinal differences among Period means (Table 1). For comparison purposes, Table 1 also shows overall mean across subclasses for Period F, although these are biologically meaningless since as shown below there were significant differences among the subclasses in this Period. Table 1. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 6 to 7 wk (Period B) and 2 to 3 wk (Period D) before initiation of egg production, and 2 to 3 wk after initiation of egg production (Period F).1   Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117    Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117  1Values for Period B and D are based on combined data of the 4 treatment subclasses, as subclasses did not differ by ANOVA. Values for Period F are for comparison purposes only, as the subclasses that were pooled differed significantly in hormone levels by ANOVA. a,b,cValues in a row that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). View Large Table 1. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 6 to 7 wk (Period B) and 2 to 3 wk (Period D) before initiation of egg production, and 2 to 3 wk after initiation of egg production (Period F).1   Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117    Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117  1Values for Period B and D are based on combined data of the 4 treatment subclasses, as subclasses did not differ by ANOVA. Values for Period F are for comparison purposes only, as the subclasses that were pooled differed significantly in hormone levels by ANOVA. a,b,cValues in a row that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). View Large In Period F, there were highly significant interaction effects for E2 and TT, and a similar tendency for P4. Therefore, detailed results for Period F are presented for E2, TT, and P4 showing the subclass means and interaction effects (Table 2). We also tested for differences among subclass means in this period. Table 2. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 2 to 3 wk after initiation of egg production (Period F), according to genetic line: B1980 or B2000, and feeding treatment: Slow (SF) or Fast (FF); n, number of birds tested per line × treatment combination; P, P-value for the interaction effects.     E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS        E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS    NS, P > 0.05; *P = 0.05; **P = 0.01; ***P = 0.001. a,b,cFor E2 and TT, values in a column that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). For P4, values that share the same letter supersdcript do not difer significanty by 1-tail t-test (P > 0.05). View Large Table 2. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 2 to 3 wk after initiation of egg production (Period F), according to genetic line: B1980 or B2000, and feeding treatment: Slow (SF) or Fast (FF); n, number of birds tested per line × treatment combination; P, P-value for the interaction effects.     E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS        E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS    NS, P > 0.05; *P = 0.05; **P = 0.01; ***P = 0.001. a,b,cFor E2 and TT, values in a column that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). For P4, values that share the same letter supersdcript do not difer significanty by 1-tail t-test (P > 0.05). View Large Time Trends in Hormone Levels Returning now to the time trends in hormone levels (Table 1), levels of E2 were moderate in Period B, with a distinct significant increase from Period B to D, and a further significant doubling from Period D (just prior to first egg) to Period F (just after first egg). This implies a functional role for E2 in the maturation and functioning of the pullet's reproductive system. Levels of TT were high in Period B, remained high in Period D, and doubled in Period F. In all 3 Periods, levels of TT were much higher on an absolute basis than levels of E2 and P4, implying a major functional role for TT in the maturation of the pullets reproductive system. Levels of P4 were very low in Periods B and D, with no change in levels from Period B to D. There was, however, a tenfold increase going from Period D to Period F. The low levels in Periods B and D accord well with lack of involvement of P4 in development of the reproductive tract, while the high levels in Period F accord with the known function of P4 in control of ovulation. Relative to the mean, standard errors of P4 in Period F were large compared to E2 and TT. This is probably due to the large diurnal fluctuations in P4 levels associated with ovulation, as the individual birds, although sampled within a rather narrow time window (9:00 to 12:00), were still not sampled at the same stage of the ovulatory cycle. As noted above, highly significant interaction effects were found for E2 and TT, with significant differences among subclass means (Table 2). Inspection of the subclass means reveals a similar pattern in both hormones: B1980 SF combination had the lowest hormone levels, B2000 SF combination had the highest levels, while the 2 FF groups had very similar levels, intermediate to the 2 SF groups. This pattern can be looked at in 2 ways: (1) From a feeding treatment standpoint, under FF the 2 genetic groups are very similar, while under SF they differ widely. (2) From a genetic standpoint, under B1980 the 2 feeding treatments are similar, while under B2000 they differ widely. Thus, from feeding treatment standpoint, B1980-FF and B2000-FF are postulated as similar. From genetic line standpoint, B1980-SF and B1980-FF are postulated as similar. Examination of Table 2 shows that B2000-FF and B1980-FF are almost identical for both E2 and TT, while B1980-FF and B1980-SF although similar, do differ somewhat, particularly for TT. Thus, Pattern (1) seems to fit the data more closely. Under either formulation, hormone levels for B2000 under SF are much higher than under FF. Turning now to P4. Although interaction effects were not significant for this hormone, examination of Table 2 shows the same pattern as found for E2 and TT: the B1980-SF subclass had the lowest hormone levels and B2000-SF subclass had the highest levels, while the 2 FF groups had very similar levels, intermediate to the 2 SF groups. In addition, the signature card of the interaction is presence of a significant difference between the B2000-SF and B1980-SF means. This is also found for P4, if we allow a 1-tail test. This seems acceptable, as we are looking at the P4 data in the light of the E2 and TT results. DISCUSSION All steroid hormones are known to increase at the onset of lay (Mobarkey et al., 2010). However, examination of Table 1 and the figures shows that each of the 3 hormones has its own pattern of development across the observed period. E2 shows low levels at start of observations, followed by consistent increase until after onset of lay. The increase appears to be strictly linear from beginning to end for all treatment groups except B2000 SF, which shows a sharp spike after entry into lay. This is consistent with known involvement of E2 in the development of the reproductive system. TT was present at very high levels from the very start of the sampling at 19 wk of age, and remained more or less level and high until onset of lay, when it increased considerably, again with a sharp spike for B2000 SF, as seen for E2. This is somewhat unexpected. Although TT is known to be involved in development of the female chicken reproductive tract, these high levels of TT appear to give this hormone a predominant role. P4 presents very low levels from start of observations until just before lay, when levels rise tenfold. This is as expected considering the critical role of P4 with LH in control of ovulation, while the very low levels of P4 until onset of lay are consistent with absence of any known developmental role for this hormone. The outstanding result of this study is the highly significant interaction effect of feeding treatment and line on E2, TT, and possibly P4 as well. Under SF, hormone levels were highest for the B2000 birds, lowest for the B1980 birds, while under FF, hormone levels were very similar for the 2 genetic lines. E2 and TT are both involved in development and functioning of the oviduct and reproductive system. Levels of these hormones were much higher for B2000 birds under SF, than under FF, resulting in statistically significant treatment × line interaction effects. Since B2000 birds under SF were also much more productive than under FF treatment, it would be reasonable to conclude that SF treatment exerts at least part of its positive effect on performance in B2000 birds by increasing levels of estrogen and TT during the period around entry into lay. It is puzzling that for all 3 hormones, levels were lowest for the B1980 birds under SF. We would have expected the B2000 FF birds to have captured this spot. Also puzzling is the positive effect of SF on P4 levels in the B2000 birds. We would not expect P4 to share control mechanisms with E2 and TT, their physiological roles are so different. Perhaps the levels of P4 are not primarily a direct effect of the feeding treatment, but a downstream reflection of the levels of E2 and TT. That is, the favorable development of the reproductive tract due to effect of feeding regime on E2 and TT also improves P4 levels. It is of interest to compare these results at the hormonal level to the results previously observed at the performance level in these birds (Eitan et al, 2014). In performance too, there was a striking interaction for Laying % and egg mass in the 16 d following onset of lay. Feeding treatment had little effect on Laying % or egg mass for the B1980 birds (70.9% and 444.2 g under SF; 64.2% and 415.7 g under FF), while there was a very powerful effect of feeding treatment for the B2000 birds (65.1% and 541.2 g under SF; 51.5% and 399.6 g under FF). This corresponds closely to what we have described as Pattern 2. Thus, we are left with a strong statement: SF increases hormone levels for E2 and TT in B2000 pullets at entry to lay, and SF improves reproductive performance of B2000 chicks at entry to lay. It is plausible that there is a causal relationship between the 2, mediated by the known involvement of E2 and TT in development of the reproductive tract of the female chicken. Thus, etiology of OFC appears to be primarily a matter of development of the reproductive tract under influence of E2 and TT. In this, it differs from etiology of EODES which appears to more a matter of control of ovulation. Interaction of hormone levels of fast- and slow-growing broiler lines with feeding treatment has been observed previously. Onagbesan et al. (2006) compared a standard broiler line (S) with an experimental dwarf breeder line (E) under restricted (R) and ad libitum feeding (A). Laying % was similar for EA, ER, and SR, but much less for SA, paralleling the results found in the first part of the present study (Eitan et al., 2014). At the hormone level, peak levels for P4 were lower for SA and ER, and higher for SR and EA. E2 levels were similar for EA and ER, but much lower for SA than for SR. Thus, here too, there was a strong treatment × line interaction at the hormone level. In a previous experiment, Onagbesan et al., (1999) compared 2 broiler lines: GL, selected for growth rate; and FC, selected for food conversion efficiency, again under restricted (R) or ad libitum (A) feeding, with respect to P4 production by ovarian granulosa cells in response to FSH, LH, and insulin-like growth factor 1 IGF-1. There were significant interaction effects on P4 production by granulosa cells in culture when stimulated by LH, FSH or IGF-1. Levels were low and similar for GLA and FCR, and high and similar for GLR and FCA. Thus, in GL, restriction increased hormone levels; while in FC, restriction decreased hormone levels. If, based on egg production, we take GL and FC to correspond roughly to B2000 and B1980, respectively, and A and R to correspond roughly to FF and SF, we find again that restriction increased hormone levels in the faster-growing lower-laying line. CONCLUSIONS The results of this study show that the OFC and EODES have different etiologies. EODES is primarily a matter of control of ovulation, while OFC is a matter of development of the reproductive tract by the steroid hormones, E2 and TT. Careful feed restriction during entry to lay, by increasing steroid hormone levels, may be a useful tool to bolster reproductive performance that has been depressed by selection for faster growth rate and higher breast percent. Notes This research was supported by the US-Israel Binational Agricultural Research and Development Fund (BARD) and by the Israel Poultry Council. REFERENCES Decuypere E., Hocking P. M., Tona K., Onagbesan O., Bruggeman V., Jones E. K. M., Cassy S., Rideau N., Metayer S., Jego Y., Putterflam J., Tesseraud S., Collin A., Duclos M., Trevidy J. J., Williams J.. 2006. Broiler breeder paradox: a project report. Worlds Poult. Sci. J.  62: 443– 453. Google Scholar CrossRef Search ADS   Eitan Y., Lipkin E., Soller M.. 2014. Body composition and reproductive performance at entry into lay of anno 1980 versus anno 2000 broiler breeder females under fast and slow release from feed restriction. Poult. Sci.  93: 1227– 1235. Google Scholar CrossRef Search ADS PubMed  Eitan Y., Soller M.. 2009. Problems associated with broiler breeder entry into lay: a review and hypothesis. Worlds Poult. Sci. J.  65: 641– 648. Google Scholar CrossRef Search ADS   FASS. 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching, 3rd edition Writing Committee Animal Handling and Transport . 3rd ed. Federation of Animal Science Societies, Champaign, IL. Hocking P. M. 1996. Role of body weight and food intake after photostimulation on ovarian function at first egg in broiler breeder females. Br. Poult. Sci.  37: 841– 851. Google Scholar CrossRef Search ADS PubMed  Hocking P. M., Gilbert A. B., Walker M., Waddington D.. 1987. Ovarian follicular structure of white leghorns fed ad libitum and dwarf and normal broiler breeders fed ad libitum or restricted until point of lay. Br. Poult. Sci.  28: 493– 506. Google Scholar CrossRef Search ADS PubMed  Jaap R. G., Muir F. V.. 1968. Erratic oviposition and egg defects in broiler-type pullets. Poult. Sci.  47: 417– 423. Google Scholar CrossRef Search ADS   Katanbaf M. N., Dunnington E. A., Siegel P. B.. 1989a. Restricted feeding in early and late-feathering chickens. 1. Growth and physiological responses. Poult. Sci.  68: 344– 351. Google Scholar CrossRef Search ADS   Katanbaf M. N., Dunnington E. A., Siegel P. B.. 1989b. Restricted feeding in early and late-feathering chickens. 2. Reproductive responses. Poult. Sci.  68: 352– 358. Google Scholar CrossRef Search ADS   Meijerhof R. 2005. The impact genetics has on breeder management. World Poult . 21: 18– 21. Mobarkey H., Avital N., Heiblum R., Rozenboim I.. 2010. The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens. Domest. Anim. Endocrinol.  38: 235– 243. Google Scholar CrossRef Search ADS PubMed  Nash J. P., Cuisset B. D., Bhattacharyya S., Suter H. C., Le Menn F., Kime D. E.. 2000. An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20 β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss). Fish Physiol. Biochem.  22: 355– 363. Google Scholar CrossRef Search ADS   Onagbesan O. M., Decuypere E., Leenstra F., Ehlhardt D. A.. 1999. Differential effects of amount of feeding on cell proliferation and progesterone production in response to gonadotrophins and insulin-like growth factor I by ovarian granulosa cells of broiler breeder chickens selected for fatness or leanness. J. Reprod. Fertil.  116: 73– 85. Google Scholar CrossRef Search ADS PubMed  Onagbesan O. M., Metayer S., Tona K., Williams J., Decuypere E., Bruggeman V.. 2006. Effects of genotype and feed allowance on plasma luteinizing hormones, follicle-stimulating hormones, progesterone, estradiol levels, follicle differentiation, and egg production rates of broiler breeder hens. Poult. Sci.  85: 1245– 1258. Google Scholar CrossRef Search ADS PubMed  Renema R. A., Robinson F. E., Zuidhof M. J.. 2008. Management and feeding of broiler breeders XXII World's Poultry Congress, Brisbane Australia, session #3, OR101. Renema R. A., Rustad M. E., Robinson F. E.. 2007. Implications of changes to commercial broiler and broiler breeder body weight targets over the past 30 years. Worlds Poult. Sci. J.  63: 457– 472. Google Scholar CrossRef Search ADS   Robinson F. E., Renema R. A., Bouvier L., Feddes J. J. R., Wilson J. L., Newcombe M., McKay R. I.. 1998. Effects of photostimulatory lighting and feed allocation in female broiler breeders. Reproductive development. Can. J. Anim. Sci.  78: 603– 613. Google Scholar CrossRef Search ADS   Robinson F. E., Robinson N. A., Hardin R. T., Wilson J. L.. 1995. The effects of 20-week body weight and feed allocation during early lay on female broiler breeders. J. Appl. Poult. Res.  4: 203– 210. Google Scholar CrossRef Search ADS   Sharp P. J. 1993. Photoperiodic control of reproduction in the domestic hen. Poult. Sci.  72: 897– 905. Google Scholar CrossRef Search ADS PubMed  Spradley J. M., Freeman M. E., Wilson J. L., Davis A. J.. 2008. The influence of a twice-a-day feeding regimen after photostimulation on the reproductive performance of broiler breeder hens. Poult. Sci.  87: 561– 568. Google Scholar CrossRef Search ADS PubMed  Van Middlekoop J. H. 1971. Shell abnormalities due to the presence of two eggs in the shell gland. Arch. fur Geflugelkd.  352: 122– 127. Van Middlekoop J. H. 1972. The relationship between ovulation interval of White Plymouth Rock pullets and the laying of abnormal eggs. Arch. fur Geflugelkd.  36l: 223– 230. Walzem R. L., Simon C., Morishita T., Lowenstine L., Hansen R. J.. 1993. Fatty liver hemorrhagic syndrome in hens overfed a purified diet. Selected enzyme activities and liver histology in relation to liver hemorrhage and reproductive performance. Poult. Sci.  72: 1479– 1491. Google Scholar CrossRef Search ADS PubMed  Yu M. W., Robinson F. E., Etches R. J.. 1992. Effect of feed allowance during rearing and breeding on female broiler breeders.: 3. ovarian steroidogenesis. Poult. Sci.  71: 1762– 1767. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Hormonal levels of estradiol, testosterone, and progesterone at entry into lay of year 1980 vs. 2000 broiler breeder females under fast and slow release from feed restriction

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0032-5791
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Abstract

ABSTRACT In the mid-1960s egg production, fertility, and hatchability of broiler breeder females dropped precipitously. Due to disrupted follicle hierarchies and development of the erratic oviposition and defective eggs (EODES) syndrome. EODES was controlled by restricting feed. In the 1990s, another set of problems arose at entry of broiler breeders into lay and characterized by high mortality followed by lower peak lay and reduction in egg and chick production. These problems are induced by even slight over-feeding, and hence we termed it the “Over Feeding Complex” (OFC). We have speculated that OFC is a quasi-EODES condition, induced by the intense selection for increased breast proportion. To test this, we compared, under fast (FF) and slow (SF) release from feed restriction, body composition and reproductive performance of a broiler breeder from year 1980 (B1980) and kept without selection for performance traits since then, to a line hatched in 2000 (B2000). During the first 16 d of lay, feeding treatment had little effect on egg mass or Laying % for the B1980 birds, while for the B2000 birds, SF treatment resulted in significantly greater egg mass and Laying % compared to FF, showing that the OFC indeed manifested in this experiment. However, contrary to hypothesis, follicle hierarchies were normal for both lines under both feeding treatments. To gain further insight into the OFC syndrome, we here report levels of estradiol, testosterone, and progesterone for these line and treatment groups in the time period leading up to and into lay. A significant line × feeding treatment interaction effect was found for estradiol and testosterone, to a lesser extent for progesterone. For all 3 hormones, for B1980 levels 2 to 3 wk post entry into lay were similar and intermediate under FF and SF, but differed significantly for B2000, being much greater under SF than under FF. Thus, the hormonal effects were parallel and may explain the egg mass and Laying % effects of FF and SF in the 2 genetic types. INTRODUCTION In the mid-1960s egg production, fertility, and hatchability of broiler breeder females dropped precipitously, endangering the economic viability of the industry. The problems were shown to be due to over-production of large yellow follicles leading to disrupted follicle hierarchies and consequent development of the erratic oviposition and defective eggs (EODES) syndrome (Jaap and Muir, 1968; Van Middlekoop, 1971, 1972). It was soon found that EODES could be successfully controlled by restricting feed intake of broiler breeder females; initially during rearing, and later during the entire reproductive period (reviewed in Decuypere et al. 2006 and Renema et al. 2007). Control of EODES by restricting feed intake, led inevitably to the self-evident hypothesis that EODES was caused by ad libitum feeding of the broiler breeder hens. This was confirmed by raising broiler breeders ad libitum in suitably designed experiments (Hocking et al., 1987; Katanbaf et al., 1989a,b; Yu et al., 1992; Walzen et al., 1993). Many studies have shown that the degree of development of EODES appears to stand in monotonic relation to the degree of over-feeding relative to breeder recommendations and ad libitum (reviewed in Eitan and Soller, 2009). In the 1990s, a new set of problems arose centered at entry of the broiler breeder into lay (Robinson et al., 1995; Hocking, 1996; Meijerhof, 2005) and characterized by high mortality at onset of lay (Spradley et al., 2008) followed by lower peak lay and reduction in egg and chick production (Katanbaf et al., 1989a,b; Robinson et al., 1998; Renema et al., 2008). This set of problems is induced by even slight over-feeding at entry to lay (so called “aggressive feeding”) relative to the breeder's recommendation, and hence we have termed it the “Over Feeding Complex” (OFC, in Eitan and Soller, 2009). We have elsewhere speculated that the OFC is a quasi-EODES condition, induced by a synergistic interaction between the very severe feed restriction of the growing pullet until the start of the maturation processes at age 15 to 16 wk, and the intense selection for increased breast proportion beginning at about 1980 (Eitan and Soller, 2009). To test this hypothesis, Eitan et al. (2014) compared body composition and reproductive performance of a broiler breeder line hatched in 1980 (B1980) and kept without selection for performance traits since then, to a broiler breeder line hatched in 2000 (B2000), under fast (FF) and slow (SF) release from feed restriction. Breast proportion increased by 40%, whereas abdominal fat pad proportion decreased by 50% when comparing B1980 and B2000 birds. Interaction effects on these traits were not present. In contrast, there was a striking interaction for Laying % and egg mass for the 2 wk following onset of lay. Feeding treatment had little effect on egg mass or Laying % for the B1980 birds, while there was a very powerful positive effect of SF feeding treatment for the B2000 birds showing that the OFC indeed manifested in this experiment. However, contrary to initial hypothesis, feeding treatment did not interact with genetic type with respect to follicle development; hierarchies were normal for B1980 and B2000 birds under both feeding treatments. In order to gain further insight into the OFC syndrome, as part of the same experiment (Eitan et al., 2014), we measured and here report hormone levels for estradiol (E2), testosterone (TT), and progesterone (P4), in the time period leading up to and across entry into lay in B1980 an B2000 birds under SF and FF treatments. These are the main plasma gonadal steroids that represent the developmental status of the gonadal axis of the hen. MATERIALS AND METHODS Population In this study, 2 female broiler breeder lines were compared under fast and slow release from restriction at entry into lay. One, representing the genetic level of the year 1980; the other, representing the genetic level of the year 2000. Effects of line and feeding treatment on body composition and reproductive performance were published previously (Eitan et al., 2014), and full description of stocks, rearing procedures, and experimental feeding treatments are provided in that publication. The B1980 group consisted of 47 chicks hatched October 29, 2001, derived from an Arbor Acre broiler female line established in 1980 and maintained since then at the North Talpiot Experimental Farm of the Hebrew University of Jerusalem, without selection for broiler performance traits. The B2000 group consisted of 41 Cobb 500 chicks hatched October 25, 2001 (Brown Yavne Hatchery, Kvutzat Yavne, Israel). Experimental Feeding Treatments Food was provided ad libitum until 21 d of age for the B2000 group and 30 d for the B1980 group. Following recommendations of the “Cobb Breeder Management Guide, 1998”, food quantities were adjusted for the B2000 to reach target weights of 1580 g at 105 d and 2400 g at 147 d. Following recommendations of the “Arbor Acre Management Guide, 1980”, feed quantities for B1980 were adjusted to reach target weights of 1580 g at 105 d and 2140 g at 147 d. At 21 wk, both groups (B2000 and B1980) were divided into 2 subgroups, and each subgroup received either a FF or a SF treatment. For the FF treatment, beginning at 70 g/d for B1980 and 50 g/d for B2000, feed quantities were increased by +20 g/d for wk 22 and +10 g/d weekly thereafter, until maximum of 140 g/d. The birds were maintained at this level until all entered lay. For the SF treatment, beginning from the same amounts of feed/d as above, feed quantities were increased by +10 g/d for wk 22 and +5 g/d weekly thereafter, until maximum of 110 g/d, at which feed level all SF pullets had entered lay. Research was done before formation of university institutional animal care and use committee, but all procedures were in accord with accepted principles and guidelines for animal care (FASS, 2010). Photoperiod Chicks were brooded for 21 d in electrically heated batteries, and then transferred to floor pens until reaching suitable BW for transfer to individual cages in a light-controlled shed at 28 to 40 d, depending on line. Naturally decreasing photoperiod was provided until transfer to cages. In the shed, photoperiod was 8L:16D until 21 wk of age. At 21 wk, photoperiod was increased to 12L:12D, and by 1 h every 2 wk until 17L:7D. Sample and Data Collection The birds were divided into 4 groups of 20 to 24 birds each, according to genetic-type × feeding-treatment combination. Each group was divided into 2 replicate blocks of 10 to 12 birds each. The birds were housed in a darkout shed, in individual cages, according to the following scheme, where groups are coded A = B1980-SF, B = B1980-FF, C = B2000-SF, and D = B2000-FF; and replicates are coded 1 and 2: A1, A2: B1, B2 etc. A1-B1-C2-D2: upper row D1-C1-B2-A2: lower row Although exact time of day when samples were collected was not recorded, it was always between 9:00 to 12:00 am. Thus, any residual diurnal variation should not affect hormone levels for E4 and TT which do not show diurnal variation. However, P4 levels are known to show large diurnal variation, and this residual variation was not controlled. Thus, it may be a source of error variation for P4. That being said, the time of collection did not vary systematically among treatment groups, and consequently should not confound the results. Hormone Assays Heparinized blood samples of 2 to 3 mL were drawn from the brachial vein at 19 wk, and weekly from 21 wk. Samples were kept in ice, and taken to the Hebrew University Faculty of Agriculture at Rehovot. Plasma was stored at –20ºC until assayed. All samples were measured on the same day, and plasma E2, TT, and P4 were measured in a single assay by enzyme-linked immunosorbent assay according to a previously described method (Nash et al., 2000), which was validated for laying hens. Dilutions of primary antibody and tracer were 1:320 000 and 1:320 for E2; 1:160 000 and 1:160 for TT and 1:5000 and 1:50 for P4. For all 3 hormones, the minimal detectable dose was 0.78 pg/mL, and the inter-assay coefficients of variation were less than 5%. Statistical Analyses Since at a given chronological age every bird may be in a different physiological state relative to entering lay, changes in the mean hormone value of a population with age primarily reflect the changing proportion of birds that are in lay or close to lay, rather than the physiological state of the bird. In order to account for this, data were plotted against each individual's “physiological age”. That is, for each bird, the week of laying the first egg was taken as the origin (zero) week for measuring physiological age. Measurement dates for each bird were assigned positive (+) or negative (–) physiological age values, according to number of weeks preceding or following week of initiation of lay (zero). As compared to chronological age, the physiological age analysis has 2 weaknesses. (1) The number of samples in each “physiological week” for a given treatment group depends on the number of birds at a given removal from first egg, and thus varies much more than when analyzed according to the chronological age. Thus, the procedure introduces a degree of experimental sampling of birds in moving from week to week. (2) Photoperiod and feeding quantities were increased systematically for all birds according to chronological age. Consequently, the birds at a given physiological age can be at a different photoperiod and feeding levels, depending on the effect of their genetic type and feeding treatment on age at first egg. It should be noted, however, that after the first week, the photoperiod for all treatment groups was above the minimal duration of 10 to 13 h of light needed for entering lay (Sharp 1993). To test for statistical significance of treatment and genetic-line effects, data were analyzed as a 2-way ANOVA with genetic line, and feeding treatment as the main effects, using the Fit Model procedure of JMP version 4.01 (SAS Institute Inc., Cary, NC). To reduce fluctuations among sample groups in the ANOVA, the data of each 2 consecutive physiological ages, starting from –9 wk were combined into “Periods” of combined weeks, i.e., Period A: –8, –9 wk; Period B: –6, –7 wk; Period C: –4, –5 wk; Period D: –2, –3 wk; Period E: –1, 0, +1 wk; Period F: +2, +3 wk. ANOVA analysis for each of the 3 hormones was implemented separately for periods B, D, and F. Results for E2, TT, and P4 are presented in Table 2. When interaction effects were significant we tested subclass means for significance of differences between line × treatment groups, and examined the obtained values for the story that they tell. The usual P = 0.05 significance level was used; correction was not made for multiple tests. RESULTS Hormone Levels by Physiological Age Figures 1A, 2 and 3 show mean hormone levels by physiological age for E2, TT, and P4. Samples were taken for all birds at the same chronological start point. The treatment groups, however, entered lay at different chronological ages (as may be seen in Figure 1A for E2). Consequently, physiological start point was further from first egg for groups with later onset of sexual maturity (e.g., start point at –11 wk for B2000 SF) and closer to first egg for groups with earlier onset (e.g., –8 wk for B1980 FF and SF). Figure 1. View largeDownload slide Mean hormone levels by physiological (A) and chronological (B) age for estradiol (E2). (A) Physiological age for estradiol (E2). (B) Chronological age for estradiol (E2). Figure 1. View largeDownload slide Mean hormone levels by physiological (A) and chronological (B) age for estradiol (E2). (A) Physiological age for estradiol (E2). (B) Chronological age for estradiol (E2). Figure 2. View largeDownload slide Mean hormone levels by physiological age for testosterone. Figure 2. View largeDownload slide Mean hormone levels by physiological age for testosterone. Figure 3. View largeDownload slide Mean hormone levels by physiological age for progesterone (P4). Figure 3. View largeDownload slide Mean hormone levels by physiological age for progesterone (P4). Figure 1A shows E2 levels against physiological age. Differences among treatment groups within age groups in the weeks prior to first egg are small, indicating good technical control of measurement variation. There is a steady slow and linear rise in E2 levels from 11 wk before lay until 2 or 3 wk after onset of lay, when the birds were taken for examinations. The steady rise continues unabated across first egg and into the laying period for all except the B2000 SF group. This group shows a marked increase on entry into lay, clearly distinguishing it from the other treatment groups. It is noteworthy that after entry into lay B2000 and B1980 differ widely under SF (B2000 SF is highest group and B1980 SF is the lowest), while under FF, B2000 and B1980 are intermediate and very similar in hormone levels. Thus, feeding-treatment effect appears to depend on line, i.e., technically speaking there is an apparent feeding × line interaction effect. E2 Levels by Chronological Age Figure 1B shows E2 levels against chronological age. The much greater differences among treatment × line groups at given chronological age (Figure 1B), compared to much smaller differences among groups at given physiological age (Figure 1A), clearly justifies the use of physiological age as our parameter of interest. Figure 2 shows TT levels against physiological age. In contrast to E2, there are large differences among treatment groups at the same physiological age and across physiological age within treatment groups, particularly for B1980 FF and B2000 FF. Differences within and across physiological age groups for B2000 SF and B1980 SF were much smaller. Overall, TT levels start high and remain high until wk 0. At this point, for B1980 FF and B1980 SF, there is tendency to increase, but not dramatically. B2000 SF, however, increased markedly from being the treatment group with lowest hormone level prior to first egg, to being the treatment group with highest hormone level after first egg. The post-lay interaction pattern seen with E2 recurs here also: under SF, B2000 has highest hormone levels, B1980 the lowest, while under FF, B2000 and B1980 are similar and intermediate. Figure 3 shows mean P4 levels of the 4 treatment groups against physiological age. Hormone levels start and remain very low until –2 wk, and rise dramatically with first egg. Here too, the very tight scatter of means of the 4 groups within any given physiological age prior to first egg shows that the assay was highly reproducible and had little technical variation. Thus, the large differences among groups on entry into lay and post entry into lay can be taken to reflect the actual variation in hormone levels, for the sampled groups. This may be due in part to residual diurnal variation within the time window of sampling, and differences among the sampled birds in the same physiological-age sample groups as explained above. Here too, the same post-lay hormone level pattern can be discerned in large part: under SF, B1980 is lowest and B2000 is highest, while under FF, B1980 is intermediate. B2000 is too variable to call. Statistical Significance of Treatment × Genetic Line Effects To test for statistical significance of treatment and genetic-line effects, data were analyzed as a 2-way ANOVA, with genetic line and feeding treatment as main effects. As noted in Methods, to reduce fluctuations among sample groups in the ANOVA, the data of each 2 consecutive physiological ages, starting from –9 wk were combined into “Periods”, running from Period A: –9, –8 wk to Period F: +2, +3 wk. ANOVA analysis for each of the 3 hormones was implemented separately for periods B, D, and F. There were only a few sporadic significant main effects, none of which remained significant after Bonferroni correction for multiple tests. For Periods B and D, interaction effects were also not significant. Therefore, for these periods, data were combined across subclasses to provide overall estimates of time trends in hormone levels and were tested for longitudinal differences among Period means (Table 1). For comparison purposes, Table 1 also shows overall mean across subclasses for Period F, although these are biologically meaningless since as shown below there were significant differences among the subclasses in this Period. Table 1. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 6 to 7 wk (Period B) and 2 to 3 wk (Period D) before initiation of egg production, and 2 to 3 wk after initiation of egg production (Period F).1   Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117    Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117  1Values for Period B and D are based on combined data of the 4 treatment subclasses, as subclasses did not differ by ANOVA. Values for Period F are for comparison purposes only, as the subclasses that were pooled differed significantly in hormone levels by ANOVA. a,b,cValues in a row that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). View Large Table 1. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 6 to 7 wk (Period B) and 2 to 3 wk (Period D) before initiation of egg production, and 2 to 3 wk after initiation of egg production (Period F).1   Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117    Period B (n = 35)  Period D (n = 35)  Period F (n = 66)    Mean  SE  Mean  SE  Mean  SE  Estradiol  0.336a  0.027  0.520b  0.039  0.815c  0.047  Testosterone  1.152a  0.043  1.122a  0.091  2.042b  0.092  Progesterone  0.089a  0.012  0.109a  0.038  1.127b  0.117  1Values for Period B and D are based on combined data of the 4 treatment subclasses, as subclasses did not differ by ANOVA. Values for Period F are for comparison purposes only, as the subclasses that were pooled differed significantly in hormone levels by ANOVA. a,b,cValues in a row that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). View Large In Period F, there were highly significant interaction effects for E2 and TT, and a similar tendency for P4. Therefore, detailed results for Period F are presented for E2, TT, and P4 showing the subclass means and interaction effects (Table 2). We also tested for differences among subclass means in this period. Table 2. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 2 to 3 wk after initiation of egg production (Period F), according to genetic line: B1980 or B2000, and feeding treatment: Slow (SF) or Fast (FF); n, number of birds tested per line × treatment combination; P, P-value for the interaction effects.     E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS        E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS    NS, P > 0.05; *P = 0.05; **P = 0.01; ***P = 0.001. a,b,cFor E2 and TT, values in a column that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). For P4, values that share the same letter supersdcript do not difer significanty by 1-tail t-test (P > 0.05). View Large Table 2. Mean plasma estradiol (E2), testosterone (TT), and progesterone (P4) levels (ng/mL) with standard error (SE) of broiler breeders tested 2 to 3 wk after initiation of egg production (Period F), according to genetic line: B1980 or B2000, and feeding treatment: Slow (SF) or Fast (FF); n, number of birds tested per line × treatment combination; P, P-value for the interaction effects.     E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS        E2  TT  P4  Line  Feed  n  Mean  SE  n  Mean  SE  n  Mean  SE  B1980  SF  15  0.816a  0.098  15  1.461a  0.193  15  0.784a  0.246    FF  18  0.962a  0.089  18  1.999b  0.177  18  1.191a,b  0.224  B2000  SF  16  1.386b  0.095  16  2.797c  0.187  16  1.321b  0.238    FF  17  0.956a  0.092  17  1.912b  0.182  17  1.212a,b  0.231    P    **      ***      NS    NS, P > 0.05; *P = 0.05; **P = 0.01; ***P = 0.001. a,b,cFor E2 and TT, values in a column that share the same letter superscript do not differ significantly by 2-tail t-test (P > 0.05). For P4, values that share the same letter supersdcript do not difer significanty by 1-tail t-test (P > 0.05). View Large Time Trends in Hormone Levels Returning now to the time trends in hormone levels (Table 1), levels of E2 were moderate in Period B, with a distinct significant increase from Period B to D, and a further significant doubling from Period D (just prior to first egg) to Period F (just after first egg). This implies a functional role for E2 in the maturation and functioning of the pullet's reproductive system. Levels of TT were high in Period B, remained high in Period D, and doubled in Period F. In all 3 Periods, levels of TT were much higher on an absolute basis than levels of E2 and P4, implying a major functional role for TT in the maturation of the pullets reproductive system. Levels of P4 were very low in Periods B and D, with no change in levels from Period B to D. There was, however, a tenfold increase going from Period D to Period F. The low levels in Periods B and D accord well with lack of involvement of P4 in development of the reproductive tract, while the high levels in Period F accord with the known function of P4 in control of ovulation. Relative to the mean, standard errors of P4 in Period F were large compared to E2 and TT. This is probably due to the large diurnal fluctuations in P4 levels associated with ovulation, as the individual birds, although sampled within a rather narrow time window (9:00 to 12:00), were still not sampled at the same stage of the ovulatory cycle. As noted above, highly significant interaction effects were found for E2 and TT, with significant differences among subclass means (Table 2). Inspection of the subclass means reveals a similar pattern in both hormones: B1980 SF combination had the lowest hormone levels, B2000 SF combination had the highest levels, while the 2 FF groups had very similar levels, intermediate to the 2 SF groups. This pattern can be looked at in 2 ways: (1) From a feeding treatment standpoint, under FF the 2 genetic groups are very similar, while under SF they differ widely. (2) From a genetic standpoint, under B1980 the 2 feeding treatments are similar, while under B2000 they differ widely. Thus, from feeding treatment standpoint, B1980-FF and B2000-FF are postulated as similar. From genetic line standpoint, B1980-SF and B1980-FF are postulated as similar. Examination of Table 2 shows that B2000-FF and B1980-FF are almost identical for both E2 and TT, while B1980-FF and B1980-SF although similar, do differ somewhat, particularly for TT. Thus, Pattern (1) seems to fit the data more closely. Under either formulation, hormone levels for B2000 under SF are much higher than under FF. Turning now to P4. Although interaction effects were not significant for this hormone, examination of Table 2 shows the same pattern as found for E2 and TT: the B1980-SF subclass had the lowest hormone levels and B2000-SF subclass had the highest levels, while the 2 FF groups had very similar levels, intermediate to the 2 SF groups. In addition, the signature card of the interaction is presence of a significant difference between the B2000-SF and B1980-SF means. This is also found for P4, if we allow a 1-tail test. This seems acceptable, as we are looking at the P4 data in the light of the E2 and TT results. DISCUSSION All steroid hormones are known to increase at the onset of lay (Mobarkey et al., 2010). However, examination of Table 1 and the figures shows that each of the 3 hormones has its own pattern of development across the observed period. E2 shows low levels at start of observations, followed by consistent increase until after onset of lay. The increase appears to be strictly linear from beginning to end for all treatment groups except B2000 SF, which shows a sharp spike after entry into lay. This is consistent with known involvement of E2 in the development of the reproductive system. TT was present at very high levels from the very start of the sampling at 19 wk of age, and remained more or less level and high until onset of lay, when it increased considerably, again with a sharp spike for B2000 SF, as seen for E2. This is somewhat unexpected. Although TT is known to be involved in development of the female chicken reproductive tract, these high levels of TT appear to give this hormone a predominant role. P4 presents very low levels from start of observations until just before lay, when levels rise tenfold. This is as expected considering the critical role of P4 with LH in control of ovulation, while the very low levels of P4 until onset of lay are consistent with absence of any known developmental role for this hormone. The outstanding result of this study is the highly significant interaction effect of feeding treatment and line on E2, TT, and possibly P4 as well. Under SF, hormone levels were highest for the B2000 birds, lowest for the B1980 birds, while under FF, hormone levels were very similar for the 2 genetic lines. E2 and TT are both involved in development and functioning of the oviduct and reproductive system. Levels of these hormones were much higher for B2000 birds under SF, than under FF, resulting in statistically significant treatment × line interaction effects. Since B2000 birds under SF were also much more productive than under FF treatment, it would be reasonable to conclude that SF treatment exerts at least part of its positive effect on performance in B2000 birds by increasing levels of estrogen and TT during the period around entry into lay. It is puzzling that for all 3 hormones, levels were lowest for the B1980 birds under SF. We would have expected the B2000 FF birds to have captured this spot. Also puzzling is the positive effect of SF on P4 levels in the B2000 birds. We would not expect P4 to share control mechanisms with E2 and TT, their physiological roles are so different. Perhaps the levels of P4 are not primarily a direct effect of the feeding treatment, but a downstream reflection of the levels of E2 and TT. That is, the favorable development of the reproductive tract due to effect of feeding regime on E2 and TT also improves P4 levels. It is of interest to compare these results at the hormonal level to the results previously observed at the performance level in these birds (Eitan et al, 2014). In performance too, there was a striking interaction for Laying % and egg mass in the 16 d following onset of lay. Feeding treatment had little effect on Laying % or egg mass for the B1980 birds (70.9% and 444.2 g under SF; 64.2% and 415.7 g under FF), while there was a very powerful effect of feeding treatment for the B2000 birds (65.1% and 541.2 g under SF; 51.5% and 399.6 g under FF). This corresponds closely to what we have described as Pattern 2. Thus, we are left with a strong statement: SF increases hormone levels for E2 and TT in B2000 pullets at entry to lay, and SF improves reproductive performance of B2000 chicks at entry to lay. It is plausible that there is a causal relationship between the 2, mediated by the known involvement of E2 and TT in development of the reproductive tract of the female chicken. Thus, etiology of OFC appears to be primarily a matter of development of the reproductive tract under influence of E2 and TT. In this, it differs from etiology of EODES which appears to more a matter of control of ovulation. Interaction of hormone levels of fast- and slow-growing broiler lines with feeding treatment has been observed previously. Onagbesan et al. (2006) compared a standard broiler line (S) with an experimental dwarf breeder line (E) under restricted (R) and ad libitum feeding (A). Laying % was similar for EA, ER, and SR, but much less for SA, paralleling the results found in the first part of the present study (Eitan et al., 2014). At the hormone level, peak levels for P4 were lower for SA and ER, and higher for SR and EA. E2 levels were similar for EA and ER, but much lower for SA than for SR. Thus, here too, there was a strong treatment × line interaction at the hormone level. In a previous experiment, Onagbesan et al., (1999) compared 2 broiler lines: GL, selected for growth rate; and FC, selected for food conversion efficiency, again under restricted (R) or ad libitum (A) feeding, with respect to P4 production by ovarian granulosa cells in response to FSH, LH, and insulin-like growth factor 1 IGF-1. There were significant interaction effects on P4 production by granulosa cells in culture when stimulated by LH, FSH or IGF-1. Levels were low and similar for GLA and FCR, and high and similar for GLR and FCA. Thus, in GL, restriction increased hormone levels; while in FC, restriction decreased hormone levels. If, based on egg production, we take GL and FC to correspond roughly to B2000 and B1980, respectively, and A and R to correspond roughly to FF and SF, we find again that restriction increased hormone levels in the faster-growing lower-laying line. CONCLUSIONS The results of this study show that the OFC and EODES have different etiologies. EODES is primarily a matter of control of ovulation, while OFC is a matter of development of the reproductive tract by the steroid hormones, E2 and TT. Careful feed restriction during entry to lay, by increasing steroid hormone levels, may be a useful tool to bolster reproductive performance that has been depressed by selection for faster growth rate and higher breast percent. Notes This research was supported by the US-Israel Binational Agricultural Research and Development Fund (BARD) and by the Israel Poultry Council. REFERENCES Decuypere E., Hocking P. M., Tona K., Onagbesan O., Bruggeman V., Jones E. K. M., Cassy S., Rideau N., Metayer S., Jego Y., Putterflam J., Tesseraud S., Collin A., Duclos M., Trevidy J. J., Williams J.. 2006. Broiler breeder paradox: a project report. Worlds Poult. Sci. J.  62: 443– 453. Google Scholar CrossRef Search ADS   Eitan Y., Lipkin E., Soller M.. 2014. 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Poultry ScienceOxford University Press

Published: Jun 1, 2018

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