Introduction

The use of growth promoters has been adopted for several decades in animal production to enhance the animals’ growth performance, increase their prime cuts yield, and decrease intramuscular fat deposition (Valenzuela-Grijalva et al. 2017). However, due to the human and animal health risks associated with the use of synthetic feed additives as growth promoters in meat production, synthetic or antibiotic growth promoters are now being rejected in many countries (Gonzalez and Angeles 2017). The dietary supplementation with phytobiotics, phytogenic feed additives, phytochemical feed additives, and herbal supplements or ingredients are now being considered as alternatives to the synthetic growth promoters (Fallah et al. 2013; Oloruntola et al. 2018a; Oloruntola et al. 2018b). The phytochemicals have antimicrobial, anti-stress, antioxidant, and immunomodulatory properties which made them potential growth promoters in animal production (Valenzuela-Grijalva et al. 2017).

Parts of various plants that are of medicinal importance had been used as supplements and or ingredient in the production of livestock to achieve various purposes of performance and health importance (Oloruntola et al. 2016c; Oloruntola and Ayodele 2017; Oloruntola 2018; Wenk 2003). Seeds and leaves of pawpaw were reported to neutraceutical and antioxidant properties (Kadiri et al. 2016). Pawpaw contains a high content of vitamin A, B, and C; papain; and chymopapain. The leaves and seeds of papaya contain 2,2-diphenyl-1-piccrlhydrazyhydrate (7.8 mg/ml and 1.0 mg/ml), phenol (424.89 mg GAE/100 g dry weight and 30.32 mg GAE/100 g dry weight), and flavonoid (333.14 mg GAE/100 g dry weight and 59.54 mg GAE/100 g dry weight), respectively (Maisarah et al. 2014). There could be interactions of the bioactive compounds in these two phytogens (pawpaw leaves and seeds), which needs to be examined (Oloruntola et al. 2018c). It was reported that medicinal plants could serve as anti-fertility agents (Nurcahyani et al. 2018). In particular, extract of papaya seeds was reported to cause various pathological changes in the reproductive organs of the animals, such as the shrunken of testis tubules, vacuolation of Sertoli cells, and abnormalities of germ cells among others (Lohiya et al. 2002). The hepatotoxic property that is dose and duration/time dependant was also reported for pawpaw by Amazu et al. (2014). Although, positive results have been reported when pawpaw plant parts are used as supplements or ingredients in animal feed (Hassan et al. 2014; Oloruntola et al. 2018a, c), pathological investigations on effects of the use of pawpaw leaves and seeds as dietary supplements or ingredients on broiler chickens are very few. Therefore, this study intended to investigate the effect of pawpaw leaf and seed meal composite mix dietary supplementation on haematological indices, carcass traits, and histopathological studies of broiler chickens.

Materials and methods

Ethical approval, experimental site, and pawpaw leaf and seed meal composite mix preparation and analysis

Prior to the commencement of this experiment, the approval for the use of animal and animal protocol was given by the Research and Ethics Committee of Agricultural Technology Department, The Federal Polytechnic, Ado Ekiti, Nigeria. Thereafter, the experiment was conducted at the Agricultural Technology Department Teaching and Research Farm. The experimental site has mean annual rainfall of 1247 mm and mean annual temperature of 26.2 °C and was situated at 437 mm above sea level; latitudes of 7° 37′ N and 7° 12′ N and longitudes of 5° 11′ E and 5° 31′ E (Oloruntola et al. 2016b).

Pawpaw leaves and ripe fruits were plucked from pawpaw trees within the premises of The Federal Polytechnic, Ado Ekiti, Nigeria. The fresh pawpaw leaves were shopped into smaller pieces with a sharp stainless knife and thereafter spread lightly on tarpaulin under a shed to air-dry for about 2 weeks, milled (100 μm size) to produce pawpaw leaf meal (PLM). Seeds were scooped out of ripped pawpaw fruits with clean human hands, washed gently with water, drained, and spread lightly on tarpaulin under a shed to air-dry for about 3 weeks. The dried pawpaw seeds were thereafter milled (100 μm size) to produce pawpaw seed meal (PSM). Three portions of PLM and one portion of PSM were mixed to form a pawpaw leaf meal and seed meal composite mix (PCM). The PCM were thereafter analysed for saponin (Brunner, 1984), tannin (Aina et al. 2012), terpenoid (Sofowora 1993), vitamin C (Benderitter et al. 1998), 2,2-diphenyl-1-piccrlhydrazyhydrate (Gyamfi et al. 1999), and ferric reducing antioxidant property (Pulido et al. 2002).

Experimental diets

Two basal diets were formulated for the starter (0 to 21 days) and grower/finisher phase (22 to 42 days) according to the recommendations NRC (1994) for broiler chicken. At each phase, the basal diets were divided into six equal portions and named diets 1 to 6. Diet 1 contained 0% PCM supplementation, diet 2 was supplemented with 0.2% PCM, while diet 3, diet 4, diet 5, and diet 6 were supplemented with 0.4, 0.6, 0.8, and 1.0% PCM, respectively. The crude protein and crude fibre of the basal diets were determined (AOAC 1995).

Birds, housing, and experimental design

Three hundred ninety-six 1-day-old Arbor Acres broiler chicks were distributed to six dietary treatments; comprising of 66 birds per treatment and 11 birds per replicate in a completely randomized design. The floor of the pen (200 × 110 cm) that housed the birds in each replicate was covered with wood shavings. The temperature of the house was maintained within 31 °C ± 2 for the first week of brooding and reduced by 2 °C after each consecutive 7 days until the house temperature was 26 °C ± 2. Twenty-three hours light was provided per day. The birds were fed ad libitum throughout the 42 days of the experiment.

Chicken slaughtering procedure, sample collection and analysis, carcass trait and internal organ evaluation, and histological study

On day 42 of the experiment, three birds per replicate were selected for sacrifice, tagged, and weighed. The birds were stunned and their jugular vein was cut with clean, sharp stainless knife. The blood was allowed to flow into EDTA bottle for hematological studies. The hematological indices values were determined within 2 h post collection as described by Shastry (1983).

After the blood collection, the slaughtered birds were de-feathered, eviscerated, and dressed. The dressed weight and dressed percentage of the slaughtered bird were estimated. Thereafter, the liver, heart, lung, bile, gizzard, spleen, and bursa were excised out with a clean surgical blade, blotted with tissue paper, weighed, and expressed as the percentage of slaughtered weight. The testes of one slaughtered bird from each replicate were also taken out. The liver and testes (one each/replicate) were separated for histological study, fixed in 10% neutral-buffered formalin, dehydrated in a graded alcohol series (70%, 90%, absolute ethanol), cleared with methyl benzoate, and embedded in paraffin wax.

Sections of 5 μm were cut and stained on glass slides with hematoxylin and eosin stain for light microscopic examination (Bancroft et al. 1996; Oloruntola et al. 2017). Stained sections were examined by light microscope and photographed using a digital camera.

Data analysis

The model: Xab = μ + αa + βab was used in this experiment. Where Xab is any of the response variables, μ is the overall mean, αa is the effect of the ith treatment (a = diets 1, 2, 3, 4, 5, and 6), and βab is the random error due to experimentation. All data were subjected to ANOVA using SPSS version 20 (SPSS 2011). Duncan multiple range tests (Duncan 1955) were used to determine the difference among means.

Results

Pawpaw leaf and seed meal composite mix and experimental diets

Table 1 shows the phytochemical composition and antioxidant properties of pawpaw leaf and seed meal composite mix, while the composition of experimental basal diets for starter and grower phases is shown in Table 2.

Table 1 Chemical composition and antioxidant properties of pawpaw leaf and seed meal composite mix
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Table 2 Composition and nutrient content of experimental basal diets
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Hematological indices

Effects of pawpaw leaf and seed meal composite mix (PCM) supplementation on hematological indices of broiler chickens are shown in Table 3. Although the white blood cell (WBC) counts of birds fed PCM supplemented diets were similar/comparable to that of the birds fed the control diet, the highest (P < 0.05) WBC counts were recorded in birds fed diets 2 and 6. The hemoglobin concentration (Hbc) and packed cell volume (PCV) of birds fed PCM supplemented diets were similar (P > 0.05) to those fed the control diet, except for those fed diet six had significantly (P < 0.05) lower Hbc and PCV.

Table 3 Effects of pawpaw leaf and seed meal composite mix (PCM) supplementation on hematological indices of broiler chickens
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Carcass and relative internal organ weight

The carcass and relative internal organ weights of the broiler chickens were similar (P > 0.05) across the various dietary treatments (Table 4).

Table 4 Effects of pawpaw leaf and seed meal composite mix (PCM) supplementation on carcass and relative internal organs (%SW) of broiler chickens
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Histology

Figures 1, 2, 3, 4, 5 and 6 show the histopathological sections of the liver. The histological hepatic cells appearances of birds fed control diet are hepatic tissues composed of hepatocytes disposed of in the sheet which are separated by sinusoids free of collections and inflammatory cells, moderate congestion of the portal vessels of polymorphonuclear cells (Fig. 1). However, the dietary PCM supplementation produced some pathological changes such as the marked proliferation of polymorphonuclear and mononuclear cells and activation of liver macrophage system, the Kupffer cells in the liver of the birds (Figs. 2, 3, 4, 5 and 6).

Fig. 1
figure 1

Representative micrograph of liver section (× 400) of broiler chicks fed 0.0 % PCM supplemented diet. This section shows the hepatic tissues composed of hepatocytes (H) disposed of in the sheet and is separated by sinusoids (S) free of collections and inflammatory cells, the portal vessels (arrow) shows moderate congestion of polymorphonuclear cells (star)

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Fig. 2
figure 2

Representative micrograph of liver section (× 400) of broiler chicks fed 0.2% PCM supplemented diet. This section shows the hepatic composed of the hepatic portal region (arrow) composed of the hepatic vessels, lymphatics, and bile duct (BD). The section shows marked proliferation of polymorphonuclear and mononuclear cells (arrow and star)

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Fig. 3
figure 3

Representative micrograph of liver section (× 400) of broiler chicks fed 0.4% PCM supplemented diet. This section shows liver parenchymal cells and marked proliferation of polymorphonuclear and mononuclear cells (star)

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Fig. 4
figure 4

Representative micrograph of liver section (× 400) of broiler chicks fed 0.6% PCM supplemented diet. This section shows the proliferation of polymorphonuclear and mononuclear cells (arrow and star), activation of liver macrophage system the Kupffer cells (arrowhead)

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Fig. 5
figure 5

Representative micrograph of liver section (× 400) of broiler chicks fed 0.8% PCM supplemented diet. This section shows activation of liver macrophage system the Kupffer cells (star)

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Fig. 6
figure 6

Representative micrograph of liver section (× 400) of broiler chicks fed 1.0% PCM supplemented diet. This section shows the proliferation of polymorphonuclear and mononuclear cells (arrow and star), activation of liver macrophage system, the Kupffer cells (arrowhead)

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The histological sections of the testes are shown in Figs. 7, 8, 9, 10, 11 and 12. In birds fed 0.0 and 0.2% PCM supplemented diets, the testicular histological sections showed coils of seminiferous tubules which contain germinal epithelium with germ cells at varying degree of maturity. The interstitium is free of collection and contained interstitial cells of Leydig (Figs. 7 and 8). However, varying histological changes such as reduces spermatogenic activities in the seminiferous tubules, congestion of seminiferous tubules, tunica albuginea and interstitial, and medial hypertrophy of the blood vessels were produced by 0.4, 0.6, 0.8, and 1.0% PCM supplementation in the broiler chickens (Figs. 9, 10, 11 and 12).

Fig. 7
figure 7

Representative micrograph of testis section (× 400) of broiler chicks fed 0.0% PCM supplemented diet. This section shows the testicular tissue composed of coils of seminiferous tubules (ST). The ST contains germinal epithelium with germ cells at varying degree of maturity (line). The interstitium (IST) is free of collection and contained interstitial cells of Leydig (arrowhead)

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Fig. 8
figure 8

Representative micrograph of testis section (× 400) of broiler chicks fed 0.2% PCM supplemented diet. This section appears as in Fig. 7 above. SPC sperm cells, ST seminiferous tubules, IC interstitial cells

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Fig. 9
figure 9

Representative micrograph of testis section (× 400) of broiler chicks fed 0.4% PCM supplemented diet. This section shows reduced spermatogenic activities in the seminiferous tubules (ST)

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Fig. 10
figure 10

Representative micrograph of testis section (× 400) of broiler chicks fed 0.6% PCM supplemented diet. This section shows reduced spermatogenic activities in the seminiferous tubules. (Line), L lumen, IC interstitial cells

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Fig. 11
figure 11

Representative micrograph of testis section (× 400) of broiler chicks fed 0.8% PCM supplemented diet. This section shows seminiferous tubules (ST), TA tunica albuginea, IC interstitial congestion

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Fig. 12
figure 12

Representative micrograph of testis section (× 400) of broiler chicks fed 1.0% PCM supplemented diet. This section appears as in Fig. 11 above. The blood vessels show medial hypertrophy (arrow)

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Discussion

Hematological indices had been recognised as one of the indicators for assessing the health status of animals (Oloruntola et al. 2016a). The immunomodulatory effect of phytogenic feed supplements or ingredients in animals had been reported (Oloruntola et al. 2016a) and the fact that WBC count recorded for birds fed PCM supplemented diets were comparable to WBC recorded in birds fed the control diet in this study may imply that the ability of these birds to fight infection, to defend their bodies against foreign organisms’ invasion, to produce and distribute/transport antibodies were not compromised by the dietary PCM supplementation. In addition, the observed comparable values recorded for hemoglobin concentration (HBc) and packed cell volume (PCV) in the birds fed the control diet and diets 2, 3, 4, and 5 indicate nutritional adequacy and improvement/stability of haematological profile with PCM dietary supplementation from 0.0 up to 0.8%. The reduction of the HBc and PCV values in birds fed diet 6 (1.0% PCM dietary supplementation) may indicate an anemic condition in the birds, possibly due to various factors among which could be impaired utilization of essential vitamins and minerals (vitamin B2, B6, and iron) needed for erythropoiesis. At excessive amount, some phytochemicals may cause a deficit in normal absorption of nutrients and in particular, may form insoluble complexes with minerals (Chukwuebuka and Chinenye 2015). However, this observation may not have predict health implication because the values recorded for HBc (8.10–9.90 g/dl) and PCV (24.53–29.75%) in the birds fed 1.0% PCM supplemented diet falls within the normal range of 7.50–13.1 g/dl (HBc) and 26.00–45.20% (PCV) reported by Mitruka and Rawnsley (1977) for healthy birds. Akintomide et al. (2018) reported that the blood parameters of cockerels fed diets containing 0 to 15% neem leaf meal were not affected, while Oloruntola et al. (2018a) also recorded a similar blood indices values between the birds fed the control diet and 5% pawpaw leaf meal inclusive diet. Therefore, it is suspected that the reduced HBc and PCV values recorded in birds fed diet 6 (1.0% PCM dietary supplementation) in the present study could not have been due to the phyto-constituents of PCM, but probably due to other factors that were not studied in this work but has notable interaction with PCM dietary supplementation at 1.0% and produced impact on the affected blood indices in the experimental birds.

An abnormal change (increase/decrease) in the relative internal organs’ weights is one of the indicators for the animals’ response to toxins in their feed (Ayodele et al. 2016). The stable relative internal organs weight of the birds across all the dietary treatment groups in this study suggests that the health status of these birds were not negatively affected by the PCM supplementation. Further studies are however required to ascertain this hypothesis. This result disagreed with an earlier report of Oloruntola et al. (2018a) who recorded changes in relative weight of liver of broiler fed 5% pawpaw leaf meal supplemented diets compared to those fed the control diet. This disparity may be due to the differences between the PCM inclusion/supplementation levels among the two experiments. By implication, higher dietary PCM levels may produce a change in the relative weight of the broiler chickens’ liver. However, a further study is required to support this hypothesis.

Among the numerous functions of the liver is the processing of nutrient absorbed from the intestine and secretion of an alkaline fluid (bile) which functions in the digestion of fat and toxic substance removal from the living organism (Oloruntola 2018). The variation of the histological appearances of the liver cells of birds fed 0.2 to 1.0% dietary PCM supplementation compared to those fed the control diet may be related to phytochemicals/anti-nutritional factors present in PCM. According to Soetan and Oyewole (2009), anti-nutrients may present some activities which though not deeply understood, but may cause histological changes in animals. Kupffer cells are principal liver cells for phagocytosis, proinflammatory cytokines production, and antigen presentation. They are the first cells to be exposed to materials absorbed from the intestine (Naito et al. 1997). Activation of the liver Kupffer cells as being observed in the liver of birds fed PCM supplemented diets in this study may indicate the support of the dietary treatment for the normal physiological status of the birds. This is because the Kupffer cells activation is required for the removal of or tolerance of pathogens as well as acute hepatic injury (Wick et al. 2002; Ishibashi et al. 2009). In addition, the activation of macrophages as observed in the liver of birds fed PCM supplemented diets in this study may indicate an occurrence of a multi-cellular healing process and matrix degradation during the process of fibrosis resolution (Marra et al. 2009).

The polymorphonuclear cells function in defense against tissue trauma and any inciting inflammatory signals among others. Therefore, the observed proliferation of polymorphonuclear cells in the birds fed PCM supplemented diets in this study may be due to the activities of anti-nutritional factors in PCM which predisposed hepatic stress in the birds.

The observed histological changes (i.e., reduces spermatogenic activities in the seminiferous tubules, congestion of seminiferous tubules, tunica albuginea, and interstitial and medial hypertrophy of the blood vessels) in testes of birds fed 0.4 to 1.0 % PCM supplemented diets in this study may be the result of activities of an active ingredient (i.e., caricacin) which is present in pawpaw seeds (one of the components of PCM), but is found to cause sterility (Kobayashi et al. 2008). This result agreed with earlier reports of Nurcahyani et al. (2018) and Julaeha et al. (2014) who observed antispermatogenic effects of papaya seed extract in mice and rats, respectively. The compound (1,2,3,4-tetrahydropyridine-3-yl octanoate) isolated from Carica papaya seed at the concentration of 12.5 ng/μl was reported to cause decreased viability, motility, and an increased abnormality of the spermatozoa in rats (Julaeha et al. 2014). Another report also revealed that an oral dose (100 mg/kg BW) of ripe pawpaw seed produced degeneration of the germinal epithelium and germ cells, vacuole presence in the tubules, and Leydig cell number reduction in male albino rats (Udoh and Kehinde 1999).

Conclusions

It could be concluded that using PCM as a phytogenic feed supplement in broiler chicken diet for the period of 42 days did not pose negative effects on the normal haemopoietic processes and carcass and internal organs development. However, the dietary PCM supplementation caused some histological changes in the liver and testis of the broiler chicken.