Effect of short photoperiod on organ growth kinetics and serum hormone profile in pullets of domestic fowl, Gallus gallus domesticus

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1 Indian Journal of Experimental Biology Vol. 39, March 200 I, pp Effect of short photoperiod on organ growth kinetics and serum hormone profile in pullets of domestic fowl, Gallus gallus domesticus D S Dandekar, R V Devkar & A V Ramachandran* Di vision of Developmental Biology and Endocrinology, Poultry Section, Department of Zoology, Faculty of Science. M S Uni versity of Baroda, Vadodara , India Received 5 February 1999; revised 30 No vember 2000 Effect of short photoperiod (SP; LD 6: 18) treatment on serum hormo ne profile and growth rate of adrenal, thyroid, ovary, oviduct, liver and lymphoid organ was studied in rearing pullets (RIR breed) of I to day 90 old. Body weight and growth index of SP pullets were lesser as compared to pullets reared under LD 12: 12. Except for ovary (recorded marginal increment ), weights and growth indices of th yroid, adrenal and oviduct decreased under SP. Weight of li ver and lymphoid organs was higher at 30 and 90 days, in SP pullets as compared to LD 12: 12. Hi stometric data suggested that the transiti on from small to big follicles was slow in ovary of SP pullets, and also reduced foll icular atresia was noted in SP pullets. Except for hi gher corticosterone level at 30 days and higher progesterone level at 30 and 60 days, rclative level s of all the hormones at all other ages were lower in SP pullets. In general, the present observations suggested intraovarian changes in pullets exposed to SP. General body and organ growth kinetics have been studied during post-hatch development, as well as, under thyroid or growth hormone excess or deficiency' 2. Many studies have focussed on histomorphology of ovary and oviduct in the post-hatch phase of development, though such studies are predominantly on adult laying hens 3A. Similarly, except for a few studies on adrenal corticosteroids and thyroid hormones in the immature stages, most of the studies on hormonal profi le are in the adult birds in relation to ovulation and oviposition 5. A continuous evaluati on of these parameters in the immature stages from the period of hatch till sexual maturity would be meaningful in understanding the temporal changes during post-hatch development and, may reveal, causal relations with initiation of egg laying as well as various features of egg laying. Moreover, the influence of different photoperiodic conditions on rearing chicks considering egg-laying aspects has not been studied. It is likely that the photoperiod induced alterati ons in attainment of sexual maturity and quantitative and qualitative aspects of egg laying, may be reflected on the changes induced by photoperiod in the immature stages and, might ultimately lead to the development of valid morpho-physiological correlates in relation to laying performance. In this respect, previous study has reported a favourable influence of *Correspondent author short photoperiod in rearing stages amounting to stepup photic schedule, on attainment of sexual maturity, egg yield and laying performance 6. Hence, in the present study an attempt has been made to evaluate the changes in body and organ weights together with changes in T 3, T 4, progesterone and corticosterone during exposure to a short photoperiod (SP) and correlate these changes with the previously observed effects on sexual maturity and laying performance. Materials and Methods Procurement and maintenance of chicks-one day old pullets of the domestic fowl Gallus gallus domesticus of Rhode Island Red (RIR) breed were procured from Model Poultry farm, Baroda, Gujarat, India. From 1 to day 90, the chicks were housed in cages (l20x75x60 cm) and placed in light proof enclosures. The chicks were fed with water ad libitum and a rationed diet [Chick mash (day 1-56) 30 g/bird/day; and grower mash (day 57 to initiation of egg lay ing; IL) 90 g/bird/day]. The light in the cages was provided by four white flourescent tubes (250 lux). Experimental protocol-a total of 72 chicks were used for the present study. They were divided into 2 groups of 36 chicks each with 12 chicks being sacrificed monthly. In group I, birds were reared under a photoperiod of LD 12: 12 for throughout periods and in group II, birds were reared under a short photoperiod (LD 6: 18) from 1 to day 90 of post hatch.,,..

2 DANDEKAR et at. : EFFECT OF SHORT PHOTOPERIOD ON ORGAN GROWTH KINETICS 23 1 i The lights were switched on for both the groups at 0700 hrs and switched off at 1900 hrs for group I at 1300 hrs for group II by an automatic timer. The temperature in the cages was regulated by supplying additional heat by using electric heaters. Overall mortality was found to be 10%. Parameters and methods of evaluation-twelve chicks were sacrificed at 30, 60 and 90 days of posthatch. Changes in body weight, thyroid, adrenal, ovary, oviduct, liver and lymphoid organs- thymus, bursa and spleen were measured gravimetrically. The birds were quickly decapitated under mild anaesthesia to avoid stress during handling. Viscera was cut open and the organs were quickly excised, blotted free of blood and tissue fluids and gravimetric evaluations were carried out using a digital pan balance (Mettler, Germany). The organs were weighed up to 0.01 mg accuracy. The absolute weights thus obtained were converted into relative weights and expressed in terms of percentage of body weight. Per day growth rate; and growth rate kinetics were calculated on the basis of absolute body weight of individuals. Radio immuno assay (RIA}-The blood samples were collected by puncturing the right jugular vein and, later centrifuged at 4000 rpm for 40 min to obtain serum. RIA for T3, T4, progesterone and corticosterone was carried out by RIA kits. RIA for total T3, T4 and progesterone was carried out by using a commercial kit from INST AR corporation, Minnesota, USA and calibrators for total T3 and T4 formed the kit. All the reagents were brought to room temperature prior to use. For assay of total T4, four uncoated tubes were labelled for total count and then the samples were run in duplicates. At the end of incubation, except for total counts tubes, all other tubes were decanted and the moisture was removed completely and counted for one minute in the gamma counter and expressed in ngidl. Maximum binding of the assay was 35% and sensitivity of the assay was 0.25 ng. Assay procedure for total T3 was same as described for total T4 except that the incubation period was 120 min. Total T3 concentration was calculated from a logit-iog representation of calibration curve and expressed as ngiml. Sensitivity of the assay was 7 ngidl and the maximum binding was 38.23%. Assay procedure for plasma progesterone was same as described for total T4 except that the volume of zero standard, standards and plasma samples were 100/lL progesterone tracer, serum blank, standard and controls were used and progesterone concentration in the sample was calculated from a logit-iog representation of the calibration curve and expressed as ngiml. Maximum binding was 42% and the sensitivity of the assay was 0.11 ngiml. Plasma corticosterone was assayed using a commercial kit from DPC, USA following solid-phase assay procedure corticosterone and corticosterone calibrators were used and the assay procedure was same as described for total T4 except that the volume of zero standard, standards and plasma samples were 100 IlL and that of 1251 corticosterone was l000/ll. All tubes were vortexed and incubated at room temperature for 20 min. Corticosterone concentration was calculated form a logit-log representation of the calibration curve, and expressed as /lgiml. Ovary of pullets were fixed in Bouin's fluid, dehydrated and embedded in paraffin. Sections of 5/l thickness were cut on a microtome and stained with haematoxylin-eosin and mounted in DPX. Histometrics of ovarian follicles was done with the help of an occulometer. A specific region in each of the sections was selected for the follicular count. Initially, a total count of follicles was made, counting the pre-ovulatory follicles (POF) and atretic follicles (AF) separately. POF on the basis of their size, were categorised as small (6-120/lm), big ( /lm) and large (> 440/lm) follicles and counted separately. All the results were statistically analysed and depicted as mean ±SE. The data was subjected to Students t test with 95% confidence level. Results Body and organ weights-the body weight of pullets of SP treatment was significantly lower than that of LD 12: 12 treated pullets at 90 days, though at 30 and 60 days it was non-significantly greater (Table 1). Weight of thyroid gland was significantly lower in SP pullets with significantly lesser growth rate at 30, 60 and 90 days. Weight of adrenal was also lower at 90 days in SP pullets. Adrenal of SP pullets, however, showed a significantly increased growth rate between 30 and 60 days and higher weight at 60 days (Table 2). Absolute and relative weights of liver and lymphoid organ of SP chicks showed increase at 30 days. However, at 90 days, absolute and relative weights of liver showed a significant decrease at 90 days, whereas absolute weights of all the.lymphoid organs showed similar weight as that of controls. (Table 1).

3 N l;.) N Table I-Effect of Short photoperiod (SP) exposure on body weight and on different organ weights of pullets [Values are mean ±SE of 12 pullets] 30 days 60 days 90 days Body weight LD 12: ± ± ± (g) SP ± ± ± 15.87" Organ weights Abs. wt. ReI. wt. Abs. wt. ReI. wt. Abs. wt. ReI. wt. Thyroid (mg) LD 12: ± ± ± ± ± ± 0.83 SP ± ± ± 1.44" ± ± 1.47 c 4.31 ± 0.49 Adrenal (mg) LD 12: ± ± ± ± ± ± 0.87 SP ± ± ± 2.01 c ± 0.70" ± 1.47 c 9.00 ± 1.09 Ovary (mg) LD 12: ± ± ± ± ± ± 1.59 SP ±5.63 c ± i.76 c ± :L 1.25 c ± ± 1.09 c Oviduct (mg) LD 12: ± ± ± ± ± ± 0.83 SP ± ± ± ± ± ± 0.79 Liver (g) LD 12: ± ± ± ± ± ± 0.17 SP 4.86 ± 0.31c ± 0.24 c 6.23 ± ± 0.23 b 9.42 ± c ± 0.68" Z 0 :; z ttl >< '"C til 0 r s:: :> ;:0 n :r: tv 8 Thymus (g) LD 12: ± ± ± ± ± ± SP 0.46 ± 0.02" ± c ± ± c 2.20± ± c Bursa (g) LD 12: ± ± ± ± ± ± SP 0.170±0.01" ± 0.01 c ± c ± c 1.37± ± Spleen (g) LD12: ± ± ± ± ± ± 0.Q25 SP ± ± c ± c 0.11 ± O.013 c ± ± 0.019" Abs. Wt.-Absolute weight; ReI. Wt-Relative weight; NLD- LD 12: 12; SP- LD 6: 18. Significant at P "<0.05; b<0.005: «

4 DANDEKAR el at.: EFFECT OF SHORT PHOTOPERIOD ON ORGAN GROWTH KINETICS 233 Overall growth rates were significantly lower in SP pullets. Ovary of SP pullets was significantly heavier at 30, 60 and 90 days with significantly reduced growth rate between 60 and 90 days (Table 2). Organ growth index represented as a ratio of organ growth rate to body growth rate showed that both thyroid and adrenal had lower overall ratios in SP pullets. Whereas the reduced ratio for thyroid was due to a conlitantly lower ratio from day 1 to 90, that of adrenal was due to a lowered initial ratio between 0 to 30 days, despite the higher ratios between 30 and 60 days and 60 and 90 days. Growth index of ovary was consistently higher, while that of oviduct was significantly lower between 60 and 90 days resulting in lowered overall ratio (Table 3). Hormonal changes-serum corticosterone (CaRT) decreased from 30 to 90 days in both LD 12:12 and SP pullets. CaRT concentration in SP chicks at 30 days was significantly greater than that of LD 12:12 chicks. However, CaRT concentration at 60 and 90 days was significantly lower in SP chicks than LD 12: 12 chicks due to a significant fall at 60 days. Between 60 and 90 days both the groups of chicks showed a gradual decline. Serum T3 and T4 levels were significantly lower in SP chicks at all the three age groups. Both T3 and T4 showed a significant decrease at 90 days in SP chicks, however, it increased at 60 days. But in the control chicks, serum T4 level remained constant throughout, T3 levels decreased. Serum progesterone titre in SP pullets decreased significantly at 60 and 90 days, whereas in pullets exposed to LD 12: 12 it decreased at 60 days followed by a significant increase at 90 days, but still lower than the level at 30 days (Table 4). Histometric observations-temporal progression from 6-30 ~m diameter follicles to ~m diameter follicles was observed 30 to 90 days in the ovary of both LD 12: 12 and SP chicks (Table 5). However, total number of follicles and relative number of follicles of various sizes were comparatively more in the ovary of SP chicks. There was also lesser number of atretic follicles. At 90 days, whereas the ovary of LD 12: 12 birds showed no follicles of 6-30 ~m range, SP ovary showed the presence of such follicles in almost the same number as seen in 30 and 60 days old ovary. Discussion SP pullets showed an overall reduced organ weights and growth kinetics, significantly during the third month though more during the first two months. This decrease was due to the significantly reduced weights and kinetics during the third month, while the same in the first two months were greater than LD 12: 12 pullets. Apparently, there was some growth retardatory influence in the third month. Rearing of chicks of domestic fowl under different photoperiodic conditions has been attempted by many workers, mainly to evaluate the effect on attainment of sexual maturity and egg laying, but none of these studies has Table 2-Effect of photoperiod exposure on growth rate [Values are mean of 12 pullets) 0-30 days days days Overall Body weight LD 12: SP Thyroid LD 12: SP Adrenal LD 12: SP Ovary LD 12: SP Oviduct LD 12: SP Liver LD 12: SP Thymus LD 12: SP Bursa LD 12: SP Spleen LD 12: SP

5 234 INDIAN J EXP BIOL, MARCH 2001 Table 3-Effect of photoperiod on growth index in LD 12: 12 and SP pullets [Values are mean of 12 pullets] 0-30 days days days Overall Thyroid LD 12: SP Adrenal LD 12: SP Ovary LD 12: SP Oviduct LD 12: SP Liver LD 12: SP Thymus LD 12: SP Bursa LD 12: SP Spleen LD 12: SP Table 4-Effect of photoperiod exposure on serum hormone and ratio of various hormones over body weight and body organs [Values are mean ±SE of 12 pullets] Parameters 30 days 60 days 90 days Corticosterone (nglml) LD 12: ± ± ± 0.42 SP 3.78 ± 0.340" 1.57 ± 0.09 h 1.36 ± 0.94" T J (nglml) LD 12: ± ± ± SP ± 0.062b ± 0.D22 b ± b T4 (~glml) LD 12: ± ± ± SP 2.59 ±0.39" 2.90 ± ± 0.06" Progesterone (ng/ml) LD 12: ± ± ± SP ± 0.029" ± a ± e T J : T4 LD 12: SP T J : corticosterone LD 12: SP Tf body wi. LD 12: SP T J : :thyroid wi. LD 12: SP T~ : corticosterone LD 12: SP T 4 : body wi. LD 12: SP T~: thyroid wi. LD 12: , SP Corticosterone:body wi. LD 12: SP Corticosterone: adrenal wi. LD 12: SP Abs. WI.-Absolute weight; ReI. Wt-Relative weight; NLD- LD 12: 12; SP- LD 6: 18. Significant at P "<0.05; b<0.005; ' <

6 DANDEKAR el at.: EFFECT OF SHORT PHOTOPERIOD ON ORGAN GROWTH KINETICS 235 E " ::1. ~o j~ 1\ E ::1. ",,0 C5~ E ::1. N 0.!?!l~ CO, ~ N N E ;:::; ::1. "'0 Eo. CI"l"':' r<l E '" EO ::1. CI"l'V c.. CI"l Cl..J 5 z reported the effect of photoperiod on organ weights and growth kinetics. The present study reveals that SP had a favourable influence on a short-term exposure (60 days), but had retardatory influence on growth and growth kinetics on long term exposure (beyond 60 days). Favourable influence of SP on organ growth was manifested in ovary (throughout) and liver (first two months). However, the growth of adrenal, thyroid and oviduct revealed a negative correlation with SP throughout. In general, the serum levels of T3, T4, CORT and progesterone showed a decrement with age in both the groups of chicks. The higher level of CORT in the first month in SP chicks is well correlated with the recorded high relative weights of liver, thymus, bursa and spleen7. Apparently, CORT had a favourabl e influence on the growth of liver and lymphoid organs during the first 30 days. This correlation is validated by the few previous reports showing increase in weight of liver and lymphoid organs during the first month 7.9. Since there was no increase in adrenal weight, it may be speculated that the metabolic clearance rate of CORT was significantly reduced under SP during the first month leading to a higher serum CORT level and a favourable growth of CORT responsive organs. Despite the increase in weight of liver and lymphoid organs in the first month in SP chicks, the weight of these organs was similar at 90 days indicating a gradual nullification of initial growth burst. This was well corroborated by the significantly reduced CORT level during the second and third months. Though, both T) and T4 levels showed age dependent decrease in both the groups of chicks, the relative levels of both T) and T4 were lower in SP chicks, more pronouncedly with respect to T3 (approximately 40%). However, the serum T4 level was significantly lower in the third month. There is no study regarding measurement of hormone levels during the period of photoperiodic manipulations in rearing chicks, except for an isolated report of Renden et al. lo showing no alteration or slight reduction in adrenal and thyroid hormone levels at differe nt ages during photoperiodic manipulations. The above study had however used shifting combinations of photoperiods, which did not conform to an experimental design involving a fixed photo-schedule as employed in the present study. The present report is therefore the first one, which projects reduced serum CORT, TJ and T4 profile due to exposure to SP and the consequent effect on the weights of these organs

7 236 INDIAN J EXP BIOL, MARCH 2001 at 90 days in SP pullets. T3 : CORT and T4 : CORT ratios were also low during third month. However, the body weight and weights of these organs were identicalor even more than those of LD 12: 12 chicks, with better growth rates. These observations tend to imply that serum titres of T3 and T4 in SP chicks during the first two months were within the optimum threshold level with no effect on growth hormone (GH) secretion/action. However, between second and third month the level of T3 and T4 was below the optimum threshold level which may result in dampened GH secretion, as correlation between GH secretion and thyroid hormone secretion has been revealed in birds ll. 12. Significantly retarded growth of body and organs during third month, could be related to reduced levels of growth promoting hormones like T3, T4, GH and CORT. Consistently lower weights and growth rates of adrenal and thyroid in SP chicks indicated SP induced slow maturation/activation of hypothalamo-hypophysial-thyroid (HHT) and hypothalamo-hypophysial-gonadal (HHG) axes which was further confirmed by the significantly lower hormone to body weight ratios with reference to thyroid and adrenal hormones. HHG axis, however, potentiated under exposure to SP from hatch, marked by significantly increased weight of ovary. This observation between 1 and 60 days may not only be due to increased stimulation of HHG axis but also by weak negative feed back action of gonadal steroids due to raised threshold level of the feed back centre. The greater follicular pool and the presence of greater number of follicles in the range of and ).lm in ovary of SP chicks revealed the presumed activation of HHG axis. As an alternative to potentiation of HHG axis, increased responsiveness/sensitivity of ovarian tissue could also be considered feasible. Total follicular pool was significantly greater during the 3 months of study in the ovary of SP pullets. There was a progressive transition from small to big and big to large category of follicles during second and third months respectively in the ovaries of both LD 12: 12 and SP pullets. However, relative number of such follicles was always lesser under SP condition. Histometric data clearly revealed that relative hierarchical progression of follicles in terms of size, was slower in SP pullets compared to LD 12: 12. A comparison of rate of transition showed that, while 3.3% of the follicles progress from small to big in the first month and 9.8% in the second month in LD 12: 12 chicks, in SP chicks, it was nil in the first month and 2.5% in the second month. Similarly in the third month, whereas 26.4% of follicles progressed from small to big and 7.5% from big to large in LD 12: 12 pullets, only 20.4% progressed from small to big and 5.7% from big to large in SP pullets. Obviously, the pace of follicular progression was slowed down due to exposure to a short photoperiod. Another aspect of clear distinction was the degree of follicular atresia which decreased from a maximum of 30% in the first month to a minimum of 5% in the third month in SP chicks while, it increased from a minimum of 3% in the first month to a maximum of 31 % in the third month in LD 12: 12 chicks. A related consequence of these changes was the presence of higher number of follicles of less than 200 ).lm size at any time in the ovary of SP chicks and, more clearly, the continued presence of follicles of 0-30 ).lm size in the ovary of SP chicks while, there was a total absence of follicles of this size range in the ovary of LD 12: 12 chicks in the third month. The higher follicular pool and persistent presence of follicles of smaller size range suggested augmented oogonial proliferation coupled with lesser degeneration of germ cells, so that more germ cells were available for recruitment into follicles. The reduced rate of atresia extended even to follicles during folliculogenesis, as noted by decreasing follicul ar atresia under SP condition. From extensive studies on mammals, androgens have been implicated as one of the factors involved in follicular atresia l3 and in this respect it can be speculated that there was reduced androgen production in the ovary of SP pullets which was corroborated to relatively higher levels of serum progesterone in SP chicks in the first and second months. The significantly reduced degree of follicular atresia and higher serum progesterone level in the third month in SP chicks, suggested increased oestrogen production. The slower rate of foll icular growth transition in SP chicks might provide them with more time for maturational changes and consequently an augmented stimulatory response to the increased hypothalamo-hypophysial output in response to a stepup photoperiod after 90 daysl4,15, resulring in faster progression through white and yellow yolky follicular \

8 DANDEKAR el al.: EFFECT OF SHORT PHOTOPERIOD ON ORGAN GROWTH KINETICS 237 hierarchy, as characteristic of hens closer to sexual maturity and oviposition 15. In this respect, our previous study reports early attainment of sexual maturity and commencement of egg laying (by 53 days) in pullets exposed to SP to a step-up photoperiod 7 Overall, our present observations extend, the already established potential of a step-up photic schedule during rearing period to increase the stimul atory component of the HHG axis at the time of switch from short to long photoperiod l4,i5, Also, it provides evidence for favourable intraovarian changes during the period of exposure to SP and consequent competence of the ovary in a quantitative and qualitative sense to respond to the increased hypothalamohypophysial output. The early initiation of egg laying and, relatively higher egg yield from these pullets as reported previousl/, can be related to the present observations during the period of exposure to SP. Acknowledgement Financial assistance by CSIR, New Delhi in the form of Research project No.37 (0820)/93, EMR II is acknowledged. References I Hayashi K, KayaJi A G & Young V R. Synergism of triodothyronine and corticosterone on muscle protein breakdown. Biochem BioplJys ACla, 883 (1986) Yadav N K & Arneja D V. Effect of altered thyroid status on body weight and feed intake of white leghorn chicks. III dian } Poull Sci, 28(3) (1993) Etches R J & Croze F. Pl asma concentration of luteinizing hormone, progesterone and corticosterone during ACTH and corticosterone induced ovulation in the hen (Callus domeslicus). Cen Comp Endocrinol, 50 (1983) Sharp P J, Dunn I C & Ccrolini S. Neuroendocrine control of reduced persistence of egg laying in domestic hens : Evidcnce for the development of photorefractorincss. } Reprod Ferlil, 94 (1992) Wilson S C & Cunningham F J. Effect of increasing day length and intermittent lighting schedules in domcstic hens on plasma concentration of luteinizing hormone (LH) and the LH response to exogenous progesterone. Cell Comp Elldocrillol, 41 (1980) Dandekar D S. PhOloelldocrille mallipulatioll: A I/ol lel paradigm for polellliatillg poullry productivily. Effecl of limed slep-up photoperiod and mild hypo or hypercorlicalism ill domeslic fowl. Ph.D thesis submitted to the M.S. University of Baroda, Baroda, India (1998). 7 Davison, T F, Freeman, B M & Rea J. Effects of continuous treatment with synthetic ACTH or corticostcrone of immature Gallus domesticus. Cen Comp Endocrinol, 59 (1985) Davison T, Scanes C G, Flack I H & Harvey S. Effect of daily injections of ACTH on growth of thc adrenal and lymphoid tissues of two strains of immature fowls. Br Poult Sci, 20 (1979) Siegel P B, Gross W B & Dunnigton E A. Effect of dietary corticosterone in young Leghorn and meat - type cockreals. Br Poull Sci, 30 (1979) Renden J A, Lien R J, Oates S S & Bilgili S F. Plasma concentration of corticosterone and thyroid hormones in broilers provided various lighting schedules. Poull Sci, 73 ( 1994) 186. II Lam S K, Harvey S, Hall T R & Spencer G S G. Somatostatin immunoneutralization stimulates thyroid functi on in fowl. } Endocrinol, 110 (1986) Decuypere E & Kuhn E R. Thyroid hormonc physiology in galliformes: Age and strain related changes in physiological control. Am Zool, 28 (1988) 40 I. 13 Knobill E & Neill J D. The physiology of reproduclioll, (Raven Press, New York) (1990) Sharp P J. Photoperiodic control of reproduction in thc domestic hen. Poull Sci, 72 (1993) Etches R 1. Reproduclion ill POlillry, (CAB Intcrnational Waltingford, Oxon OX 108 DE, U.K.) 1996, 1.