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Production of lambs’ resilience to Haemonchus contortus
Small Ruminant Research 167 (2018) 110–116
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Production of lambs’ resilience to Haemonchus contortus Ridi Arif
a,b
b
c
c
, Fadjar Satrija , Adi Winarto , Arief Boediono , Wasmen Manalu
c,⁎
T
a
Graduate Student of Animal Physiology and Pharmacology, Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia Department of Animal Diseases and Veterinary Health, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia c Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lamb resilience Haemonchus contortus Intrauterine programming Gonadotropin
Haemonchus (H.) contortus infection in sheep is a major health problem in tropical and sub-tropical regions that cause great economic losses. Our previous studies have shown that improved uterine environments during pregnancy can improve postnatal growth and health performance of the offspring, indicated by lower mortality and morbidity. In the present experiment, we evaluated the resistance and resilience to H. contortus of lambs born to ewes injected with pregnant mare’s serum gonadotropin (PMSG) prior to mating. Improvement of the uterine environment was conducted by increasing endogenous secretion of estrogen and progesterone as pregnancy hormones during pregnancy by injecting the ewes with PMSG prior to mating. A total of 16 lambs, regardless of sex, at the age of 5 months were assigned into a 2 × 2 factorial experiment with 4 replications. The first factor was PMSG injection, consisting of two levels, i.e., lambs born to ewes without PMSG injection (NonPMSG lambs) and those born to PMSG-injected ewes (PMSG lambs). The second factor was the infection of lambs with H. contortus at the age of 5 months, consisting of two levels, i.e., lambs without infection (Non-infected lambs) and lambs infected with H. contortus (Infected lambs). Non-infected lambs were administered with distilled water in a capsule without infective larvae. Infected lambs were individually infected with a single dose containing 1200 L3 of H. contortus. Compared to non-PMSG lambs, PMSG lambs tended to have better prenatal growth indicated by greater birth weights (P = 0.06). The improved prenatal growth during pregnancy improved the postnatal growth and health performance of the lambs. Three months after infection of H. contortus, non-PMSG lambs and PMSG lambs had similar worm counts. However, the PMSG lambs showed significantly higher resilience to H. contortus as indicated by the lower fecal egg counts 6–10 weeks after infection. The higher resilience of the PMSG lambs was shown by the positive growth rate during infection, while non-PMSG lambs had a negative growth rate after infection. Prior to infection, PMSG lambs showed a higher segmented neutrophil percentage with lower lymphocyte numbers. Three months after infection, PMSG lambs had significantly higher lymphocyte and thrombocyte numbers as well as mean corpuscular hemoglobin concentration (MCHC) with lower neutrophil and monocyte numbers. The conclusion of this study is that the improvement of the uterine environment during pregnancy could be used to produce superior offspring with high resilience to the infection of H. contortus.
1. Introduction Parasite infestation by nematode in the intestine is a common problem in the animal husbandry industry. The infection of this parasite increases the operation costs and causes economic losses (Sackett et al., 2006). In addition, the problem is worse with the report that some intestinal parasites have increased resistance due to inappropriate and uncontrolled use of anthelmintics (Luffau et al., 1990). One of the main
⁎
gastrointestinal parasites in sheep is the nematode worm of Haemonchus (H.) contortus. H. contortus infection causes decreased body weight and anemia (Strain and Stear, 2001). In the case of chronic infection, the decreases in body weight and hematocrit levels are not normally found as compared to the non-infected animals. In hyper-acute cases, the infected animals can die quickly due to the serious bleeding in the gastrointestinal tract (Roberts and Swan, 1982). Several experiments have been conducted to map factors increasing
Corresponding author. E-mail address: [email protected] (W. Manalu).
https://doi.org/10.1016/j.smallrumres.2018.08.016 Received 7 February 2018; Received in revised form 20 June 2018; Accepted 20 August 2018 Available online 23 August 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
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contortus (infected lambs). The lambs in the infected lambs group were individually infected with a single dose containing 1200 L3 of H. contortus (modified from Ginting et al., 1999), while the non-infected group was administered with distilled water in a capsule without H. contortus. The experiment was conducted according to the National Institute of Health’s guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978).
the resistances of animals to H. contortus. One of the factors that has become a popular topic in veterinary parasitology is the genes that control the resistance phenotype (Alba-Hurtado and Munoz-Guzman, 2013). However, it is known that the genetically resistance mother does not always produce resistant offspring because the inheritance of the resistance phenotype to the offspring is not absolute (Marshall et al., 2013). Therefore, crossing with a strict selection can be used to produce offspring having high resistance. However, the method takes longer and requires strict record keeping (Wanyangu et al., 1997). Therefore, a more efficient new approach or method is required to optimize and increase the expression of the resistance genotype in offspring. Based on the understanding that the resistance phenotype is a form of united and coordinated function contributed by some bodily functions, to optimize and activate the resistance phenotypes, the improvement of all body systems is required. One approach is to optimize the development of the organ systems during the growth and development of the embryo and fetus during pregnancy. Improvement of growth and development of the embryo and fetus during pregnancy can be achieved by increasing the signals and factors controlling the growth and development of the uterine and placenta (Fowden et al., 2008; Fowden and Forhead, 2009a) by optimizing the availabilities of pregnancy hormones during pregnancy. Improved uterine and placental structure and functions during pregnancy improve gene expression and protein abundance that eventually function as epigenetic signals that affect fetal growth and development as well as phenotype diversity, which affects postnatal growth and physiological variability (Fowden and Forhead, 2009b). Maternal serum progesterone concentrations, as the main pregnancy hormones, can be increased by gonadotropin injection of the mothers prior to mating that eventually improves prenatal and postnatal growth in sheep and swine (Manalu et al., 2000; Manalu and Sumaryadi, 1998; Rayer et al., 2015a, b; Mege et al., 2006). The improved postnatal growth also associates with the increased survival as indicated by the lower morbidity and mortality (Rayer et al., 2015a, b; Mege et al., 2006). Based on these phenomena, the present experiment was designed to evaluate the resistance and resilience to H. contortus of lambs born to ewes injected with pregnant mare's serum gonadotropin (PMSG) prior to mating. PMSG injection prior to mating increases endogenous secretion of pregnancy hormones that improve the uterine environment during pregnancy. Our previous studies have shown that PMSG-injected does prior to mating had greater uterus morphometric and histometric parameters that correlated well with the fetal weight at the end of embryonic stage of pregnancy (Arif et al., 2018). This is the first report on the improvement of lambs’ resistance and resilience to H. contortus by injecting the mother with PMSG prior to mating to improve the growth and development of the uterus and placenta that will support the prenatal growth of the offspring during pregnancy.
2.2. Experimental animals To produce lambs used in the experiment, all maternal ewes were injected with prostaglandin F2 alpha (PGF2α) two times with an 11-day interval to synchronize the estrus cycle prior to mating. The PMSGinjected ewes were injected with PMSG at a dose of 7.5 IU/kg body weight (BW) at the same time as the second PGF2α injection (Andriyanto et al., 2017). The non-PMSG-injected ewes were injected with 0.7% NaCl solution. At the estrus period, all experimental ewes were mated naturally by mixing with selected male sheep for 1 week. The experimental ewes were maintained until parturition. At parturition, the birth weights of the lambs were measured. At weaning time at the age of 20 weeks postpartum, the lambs were selected for infection of infective H. contortus larvae. Prior to infection, the experimental lambs were raised intensively in cages with the maternal ewes. During the treatment, the experimental lambs were maintained intensively in cages. 2.3. Production of infective H. Contortus larvae, artificial infection, and collecting samples Two donor sheep were prepared for production of infective H. contortus larvae. The donor sheep were administered anthelmintics for 3 consecutive days. The number of eggs in each gram of feces was measured, and the sheep were free of worm infection. The donor sheep were acclimatized for 1 week. The donor sheep were fed with grass and nongrass forage feed free of worm eggs and larvae. The mature female H. contortus worms were collected from the abomasum of sheep from the local slaughterhouse. The mature worms were collected, grinded to obtain eggs, and then cultured and nourished. After 1 week of culturing, the collection of L3 larvae was conducted. The collected L3 larvae were put into a soft capsule for further use to infect the donor experimental sheep. Two weeks after infection, the number of eggs per gram of feces was counted to ensure that the donor sheep were infected with H. contortus, and the eggs were found in the feces. The feces of the donor sheep was cultured for further collection of L3. The dose of L3 infection was estimated by calculating the concentrations, i.e., the number of L3 larvae per milliliter of distilled water. The number of infected L3 larvae was 1200 L3 larvae per experimental lamb (modified from Ginting et al. (1999).
2. Materials and methods 2.4. Infection of experimental lambs 2.1. Experimental design The experimental lambs were infected with infective larvae packed in a capsule directly into the stomach using a stomach tube. The uninfected lambs were administered with distilled water in a capsule without infective larvae. After infection, the fecal samples were collected weekly up to 13 weeks post-infection to measure the number of worm eggs in the feces or fecal egg count (FEC). Thirteen weeks after infection, the fecal samples were collected for 24 h to measure the total worm eggs to calculate female worm fecundity. At the end of experiment (13 weeks post infection), the blood samples were also collected to measure blood cell parameters (erythrocyte, leukocytes, and thrombocytes). After collecting blood samples, the infected lambs (nonPMSG lambs and PMSG lambs) were sacrificed to obtain the abomasum for measurement of the number of male and female worms, total number of male and female worms, and the length of male and female worms in the mucosa of the abomasum. Since the non-infected lambs
An experiment was designed in a completely randomized fashion with 2 × 2 factorial arrangement, and each experimental unit used 4 lambs. The experiment used 8 lambs born to PMSG-injected ewes (PMSG lambs) and 8 lambs born to non-PMSG injected ewes (nonPMSG lambs). The age of the experimental lambs at the beginning of experiment was 5 months, and there was no sexual grouping of the experimental lambs. The first factor was the PMSG injection, consisting of two levels, i.e., lambs born to ewes without PMSG injection prior to mating (non-PMSG lambs) and lambs born to PMSG-injected ewes prior to mating to improve endogenous secretions of pregnancy hormones to optimize the uterine environment during pregnancy (PMSG lambs). The second factor was the infection of the experimental lambs with H. contortus, consisting of two levels, i.e., lambs without infection H. contortus as a control (non-infected lambs) and lambs infected with H. 111
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2.6. Data analysis
Table 1 The number of male worms, female worms, total number of male and female worms, the length of male worms, the length of female worms, the number of worm eggs in 24 h, and the fecundity of female worms in the abomasum of nonPMSG lambs and PMSG lambs 3 months after infection with Haemonchus contortus. Variables
Non-PMSG lambs
The number of male worms (worms) The number of female worms (worms) Total number of worms (worms) The length of male worms (cm) The length of female worms (cm) The number of eggs in 24 hours (eggs) The fecundity of female worms (eggs/worm)
14.20 ± 3.30 3.80 ± 1.41
The collected data were analyzed with analysis of variance (SPSS 16). The significant effects of main factor (PMSG injection of the ewes and infection of the experimental lambs) and their interactions were tested. The significant differences between experimental units were further tested by the Duncan ranges test (SPSS 16) using α = 0.05 as a level of significance.
PMSG lambs
a
14.25 ± 4.57
a
4.00 ± 2.16
a
a
3. Results 18.00 ± 6.36 a 1.39 ± 0.08 a 1.80 ± 0.31 a 2143.13 ± 424.26 563.98 ± 298.56 a
a
18.25 ± 5.91 a 1.42 ± 0.05 a 1.34 ± 0.86 a 1885.00 ± 523.26
During the 4-week period after infection, there was no FEC found in the feces of infected lambs (Fig. 1). The number of FEC increased with time 5 weeks after infection and reached the peak level 9 weeks after infection (P < 0.05). Five weeks after infection, PMSG lambs had numerically lower FEC compared to non-PMSG lambs (P = 0.282). PMSG lambs had consistently lower FEC compared to non-PMSG lambs 6–9 weeks after infection (P < 0.05) (Fig. 1). During the period of 6, 7, 8, and 9 weeks after infection, PMSG lambs consistently had lower FEC by 51.85, 57.58, 51.61, and 38.89%, respectively, compared to non-PMSG lambs (P < 0.05). Then, 10–13 weeks after infection, the FEC of nonPMSG lambs decreased slightly with the same concentrations with those of PMSG lambs (P > 0.05). Although there was a significant decrease in the FEC of PMSG lambs 6–9 weeks after infection, the number and morphometric measurement of adult worms in the abomasum of the lambs 13 weeks after infection did not show significant differences (Table 1). The number of male worms, female worms, and the total number of worms, the lengths of male and female worms, the number of eggs in 24 h, and the fecundity of female worms in the abomasum of non-PMSG lambs and PMSG lambs 13 weeks after infection did not show significant differences (Table 1) (P > 0.05). However, there was still a tendency of numerically shorter length of female worms, lower number of eggs in 24 h, and lower fecundity and fertility of female worms in the PMSG lambs compared to non-PMSG lambs (Table 1). Prior to infection of L3 H. contortus at the weaning period, the blood parameters of hemoglobin, leukocytes, thrombocytes, hematocrit, erythrocytes, MCV, MCH, MCHC, eosinophils, band neutrophils, segment neutrophils, lymphocytes, and monocytes in all groups of experimental lambs were similar (P > 0.05) (Table 2). The similar erythrocyte parameters in PMSG and non-PMSG lambs indicated that both groups of experimental lambs had similar normal physiological conditions at the weaning period before infection. The basophils were not detected in non-PMSG and PMSG lambs. Even though there was no statistical significance in leukocyte parameters in all groups of experimental lambs
a
471.25 ± 120.29 a
a,b
Different superscripts in the same row indicate a significant difference (p < 0.05).
did not have infection, the non-infected lambs were not sacrificed. Therefore the data in Table 1 and Fig. 1 only reflects PMSG and nonPMSG lambs infected with H. contortus. Body weights of the experimental lambs were measured at lambing, 4 months postpartum (1 month before infection), 7 months postpartum (2 months after infection), and 8 months postpartum (3 months after infection).
2.5. Parameters measured The parameters measured in this experiment were the blood parameters (hemoglobin, leukocytes, thrombocytes, hematocrit, erythrocytes, average red blood cell size (MCV), hemoglobin amount per red blood cell (MCH), mean corpuscular hemoglobin concentration MCHC, basophils, eosinophils, band neutrophils, segment neutrophils, lymphocytes, and monocytes) of the experimental lambs before treatment (at the age of 5 months) and at the end of experiment (13 weeks post infection). Hematology parameters were measured by a hematology analyzer (Sysmex XP-300®). FEC was measured by the McMaster method (Roepstorff and Nansen 1998) in Laboratory of Helminthology of the Faculty of Veterinary Medicine of Bogor Agricultural University. The total number of worms (male and female) was counted manually. The length of the worms was measured using a caliper. Body weights of the experimental lambs were measured using a digital scale.
Fig. 1. Fecal egg count (FEC) of non-PMSG lambs (■) and PMSG lambs (♦)3 months after infection of Haemonchus contortus. a,b Different superscripts in the same weeks after infection indicate a significant difference (p < 0.05). 112
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Table 2 Red blood cell, thrombocyte, and leukocyte profiles of non-PMSG lambs and PMSG lambs before infection with Haemonchus contortus. Variables
Hemoglobin (g/dL) Leukocytes (103xμL) Thrombocytes (103xμL) Hematocrit (%) Erythrocytes (106xμL) MCV (fl) MCH (fg) MCHC (%) Basophils (%) Eosinophils (%) Band Neutrophils (%) Segmented Neutrophils (%) Lymphocytes (%) Monocytes (%)
Non-PMSG lambs
PMSG lambs
Level of significance (p value)
Non-Infected
Infected
Non-Infected
Infected
PMSG
Infection
Interaction
12.53 ± 0.26 9.55 ± 1.00 309.75 ± 62.30 37.50 ± 0.58 4.15 ± 0.06 90.00 ± 0.00 30.00 ± 0.00 33.25 ± 0.50 0.00 ± 0.00 0.50 ± 0.58 1.50 ± 1.00 68.75 ± 2.75 24.50 ± 2.08 4.75 ± 1.50
13.14 ± 0.54 11.78 ± 2.90 379.80 ± 70.67 40.20 ± 1.50 4.38 ± 0.22 89.80 ± 0.82 30.00 ± 0.00 33.20 ± 0.58 0.00 ± 0.00 0.80 ± 0.50 1.40 ± 1.00 69.60 ± 1.83 22.40 ± 4.19 5.80 ± 1.73
13.43 ± 1.56 12.38 ± 4.95 354.25 ± 26.89 40.25 ± 4.76 4.43 ± 0.50 90.00 ± 0.84 30.00 ± 0.00 33.50 ± 0.45 0.00 ± 0.00 0.75 ± 0.45 1.50 ± 0.89 77.00 ± 8.35 16.75 ± 6.11 5.50 ± 1.79
13.30 ± 1.24 12.85 ± 3.94 377.50 ± 37.50 39.75 ± 3.50 4.43 ± 0.40 89.75 ± 0.50 30.00 ± 0.00 33.50 ± 0.58 0.00 ± 0.00 0.25 ± 0.50 0.25 ± 0.50 75.25 ± 6.08 19.25 ± 5.25 5.00 ± 1.41
0.337 0.297 0.411 0.477 0.371 0.939 – 0.300 – 0.552 0.200 0.027 0.036 0.975
0.652 0.461 0.083 0.496 0.517 0.492 – 0.923 – 0.690 0.137 0.874 0.933 0.735
0.498 0.630 0.363 0.327 0.517 0.939 – 0.923 – 0.127 0.200 0.648 0.342 0.347
affected the segmented neutrophil percentages (P < 0.01). However, there was a significant interaction effect of infection and PMSG injection on segmented neutrophil percentages (P < 0.01) (Table 3). In PMSG lambs, infection significantly decreased segmented neutrophil percentages (P < 0.05). However, in non-PMSG lambs, infection did not affect the percentage of segmented neutrophils (P > 0.05). The lymphocyte percentage profiles of experimental lambs was similar to that of segmented neutrophils, in which there was a significant effect of infection (P < 0.01) and interaction between the PMSG injection and infection (P < 0.01) without a significant effect of PMSG injection (P > 0.05) (Table 3). In the non-PMSG lambs, infection did not change the lymphocyte percentage (P > 0.05). However, in PMSG lambs, infection increased the lymphocyte percentage (P < 0.05), while it decreased the segmented neutrophil percentage (P < 0.05). Different from lymphocyte profiles, and similar to the profiles of hemoglobin, hematocrit, and erythrocytes, infection of non-PMSG and PMSG lambs decreased the monocyte percentage observed 13 weeks after infection (Table 3). PMSG injection did not affect monocyte concentrations, and there was no interaction of infection and PMSG injection on monocyte percentages (P > 0.05) (Table 3). Regardless of PMSG injection, infection tended to decrease thrombocyte concentrations observed 13 weeks after infection (P = 0.054). Regardless of infection, PMSG and non-PMSG lambs had similar thrombocyte concentrations (P > 0.05) (Table 3). There was no interaction of infection and PMSG injection on thrombocyte
prior to infection, the PMSG lambs showed a tendency of a higher number of leukocytes. However, prior to infection, the percentage of lymphocytes in the non-PMSG lambs were higher compared to those of PMSG lambs (P < 0.05), and the percentage of segmented neutrophils in PMSG lambs were higher compared to non-PMSG lambs (P < 0.05). All these blood parameters were similar in infected and non-infected lambs. However, 3 months after infection, changes in blood parameters were observed (Table 3). In general, all groups of experimental lambs had similar leukocyte, MCV, MCH, basophil, eosinophil, and band neutrophil parameters (P > 0.05). No basophils were observed in any experimental lambs in this experiment. The percentages of MCHC in the experimental lambs were higher compared to non-PMSG lambs (P < 0.05) (Table 3). Infection significantly decreased hemoglobin concentrations, hematocrit percentages, and erythrocyte concentrations regardless of PMSG injection (P < 0.01). However, regardless of infection, PMSG lambs had similar hemoglobin concentrations, hematocrit percentages, and erythrocyte concentrations (P > 0.05) (Table 3). There was no interaction between infection and PMSG injection on hemoglobin concentrations, hematocrit percentage, and erythrocyte concentrations of the experimental lambs (P > 0.05). The profile of segmented neutrophils was different from that of band neutrophils. PMSG injection and infection did not affect band neutrophil percentages (P > 0.05). In contrast, infection significantly
Table 3 Red blood cell, thrombocyte, and leukocyte profiles of non-PMSG lambs and PMSG lambs 3 months after infection with Haemonchus contortus. Variables
Hemoglobin (g/dL) Leukocytes (103xμL) Thrombocytes (103xμL) Hematocrit (%) Erythrocytes (106xμL) MCV (fl) MCH (fg) MCHC (%) Basophils (%) Eosinophils (%) Band Neutrophils (%) Segmented Neutrophils (%) Lymphocytes (%) Monocytes (%) a,b
Non-PMSG lambs
PMSG lambs
Level of significance (p value)
Non-Infected
Infected
Non-Infected
Infected
PMSG
Infection
Interaction
13.14 ± 1.56 11.78 ± 4.95 379.80 ± 26.89 40.20 ± 4.76 4.38 ± 0.50 89.80 ± 0.84 30.00 ± 0.00 33.20 ± 0.45 0.00 ± 0.00 0.80 ± 0.45 1.40 ± 0.89 69.60 ± 8.35 b 22.40 ± 6.11 b 5.80 ± 1.79
11.56 ± 0.86 11.84 ± 1.39 269.80 ± 100.08 34.60 ± 2.30 3.84 ± 0.27 90.00 ± 0.71 30.00 ± 0.00 33.60 ± 0.55 0.00 ± 0.00 0.40 ± 0.55 1.20 ± 0.84 70.40 ± 2.41 b 24.40 ± 2.19 b 3.60 ± 0.89
14.07 ± 0.53 14.87 ± 2.90 378.00 ± 38.05 42.00 ± 1.41 4.67 ± 0.17 89.75 ± 0.96 30.00 ± 0.00 33.75 ± 0.50 0.00 ± 0.00 0.50 ± 0.58 1.00 ± 1.15 78.75 ± 2.50 a 14.75 ± 3.86 c 5.00 ± 1.83
11.92 ± 0.44 10.55 ± 3.16 361.25 ± 55.05 36.00 ± 1.15 3.97 ± 0.15 90.00 ± 0.00 30.00 ± 0.00 34.00 ± 0.00 0.00 ± 0.00 1.25 ± 0.95 2.25 ± 1.71 59.50 ± 6.35 c 34.50 ± 4.04a 2.50 ± 0.58
0.195 0.584 0.159 0.272 0.179 0.944 – 0.040 – 0.381 0.563 0.748 0.560 0.171
0.002 0.206 0.054 0.001 0.001 0.529 – 0.144 – 0.574 0.355 0.004 0.000 0.003
0.560 0.194 0.144 0.888 0.607 0.944 – 0.727 – 0.079 0.208 0.002 0.001 0.823
Different superscripts in the same row indicate a significant difference (p < 0.05). 113
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Table 4 Body weights of non-PMSG lambs (control) and PMSG lambs at lambing (birth weight), 1 month before infection (at the age of 4 months), and 2 and 3 months after infection with Haemonchus contortus. Time
Non-PMSG lambs
Non-infected Birth weight (kg) Body weight (kg) 1 month preinfection 2 months postinfection 3 months postinfection a,b
1.15 ± 0.11 15.42 ± 4.13
PMSG lambs
Infected
b
19.90 ± 4.41ab 21.43 ± 5.19ab
1.2 ± 0.05 16.16 ± 1.23
b
15.74 ± 1.98b 14.9 ± 1.77b
Level of significance (p value)
Non-infected
Infected
2.67 ± 0.26 20.70 ± 2.08a
2.19 ± 0.96 17.55 ± 2.79
24.96 ± 3.52a 28.33 ± 5.90a
19.42 ± 4.08ab 22.15 ± 5.25ab
ab
PMSG
Infection
Interaction
0.066 0.071
0.619 0.112
0.691 0.254
0.032* 0.024*
0.043* 0.032*
0.942 0.991
Different superscripts in the same row indicate a significant difference (p < 0.05).
PMSG lambs had only 230 eggs/g of feces while the non-PMSG lambs had 620 eggs/g of feces. The FEC has higher correlation with total worm counts (Roberts and Swan, 1981). The pattern of increase in the number of worm eggs per gram of feces was slightly slower in the PMSG lambs compared to non-PMSG lambs even though it further decreased and met the same level 12 weeks after infection. Previous studies have reported that the decrease in egg numbers per gram of feces generally occurred 14 weeks after infection and was followed by the constant values of eggs per gram of feces (Wanyangu et al., 1997). Since the worm number was not different, the lower number of eggs per gram of feces in the PMSG lambs indicates that the PMSG lambs have capacities to inhibit or depress the re-productivity and productivity of H. contortus worms. This assumption was based the results of blood parameters (Table 2). The improved quality of the immune system in the PMSG lambs that was shown by the higher number of eosinophils eventually decreased egg production by H. contortus. Observation of the number of worms showed that there was no difference between non-PMSG lambs and PMSG lambs. Even though the number of adult female worms was similar, the productivity of the adult female worms in the PMSG lambs was decreased. The decreased fecundity in adult female worms is highly correlated with the length of the adult female worms (Rowe et al., 2008). The numerically shorter length of adult female worms obtained from the PMSG lambs could be related to the decreased fecundity of female H. contortus worms. The higher number of lymphocytes observed in the PMSG lambs 3 months after infection may have contributed to the inhibition of worm fecundity. The higher number of lymphocytes, the increased activity of Thelper 2 (Th2) cells, the higher number of eosinophils in the abomasum, the increased number of mast cells, the increased concentrations of interleukin (IL)-4, IL-13, immunoglobulin (Ig)G, and IgA were shown to be good resistance features (Smith and Christie, 1979; Strain and Stear, 2001; Lacroux et al., 2006). The treatment could improve the immune response against gastrointestinal parasites that could influence the resilience of the lambs. The conditions of experimental lambs prior to infection were similar except that PMSG lambs had higher segmented neutrophil and lower lymphocyte percentages. These phenomena indicate the potency of higher immune system in PMSG lambs. Therefore, PMSG lambs had a higher ratio of segmented neutrophils and lymphocytes, indicating the physiological immune systems of PMSG lambs were more optimum. In general, the blood cell parameters observed in the experimental lambs prior to infection at the weaning period were similar to those reported in the region of Southeast Asia (Ginting et al., 1999). The similar decrease in the erythrocyte and leukocyte parameters of both groups of experimental lambs after infection strongly confirmed that infection was successful and produced pathological conditions (Rowe et al., 2008) in the experimental lambs. In addition, the average time required of 4 weeks from the artificial infection until the presence of worm eggs in the feces is similar to that reported previously (Vanimisetti, 2003).
concentrations (P > 0.05), indicating that PMSG lambs showed lower decreases in thrombocyte concentrations in response to H. contortus infection. The most important response to be highlighted related to the infection of H. contortus worms is the decrease in body weight of the infected lambs (Table 4). At birth, PMSG lambs had numerically higher birth weight (P = 0.066), and lambs in the group of infection had similar birth weights. One month prior to infection (at the age of 4 months), the same pattern of body weight in the experimental lambs was similar to the birth weight, in which PMSG lambs tended to have higher body weights compared to non-PMSG lambs (P = 0.071). Infected lambs had similar body weights 1 month before infection (P > 0.05). However, 2–3 months after infection, PMSG injection and infection significantly affected body weights of the experimental lambs. There was no interaction of PMSG injection and infection on the body weight of experimental lambs. PMSG lambs had significantly higher body weights compared to non-PMSG lambs during the period of 2 and 3 months after infection. Two months post-infection, infected PMSG lambs had 23.38% higher body weights compared to infected non-PMSG lambs, indicating that PMSG lambs had better growth response to infection compared to non-PMSG lambs. In the same period, non-infected PMSG lambs had a 25.43% higher body weight compared non-infected non-PMSG lambs, indicating the PMSG lambs had a significantly (P < 0.05) higher growth rate during the post-weaning period. Three months post-infection, infected PMSG lambs had a 48.66% higher body weight compared to infected non-PMSG lambs. In the same period, non-infected PMSG lambs had a 32.20% higher body weight compared to non-infected non-PMSG lambs, indicating that the PMSG lambs had better growth rates during the post-weaning period (P < 0.05). Regardless of PMSG injection, infection decreased body weight by 21.81% in PMSG lambs and 30.47% in non-PMSG lambs. This experiment showed that the improved resilience of PMSG lambs as indicated by the blood parameters responses could maintain the normal body functions as indicated by the increased body weight by 26.21% 3 months after infection compared to that 1 month prior to infection. In contrast, non-PMSG lambs had a negative growth rate (decreased body weight by 7.80%) 3 months after infection compared to that 1 month prior to infection. As a comparison, during the same period of measurements, non-infected PMSG lambs and non-infected non-PMSG lambs had 36.86% and 38.98% higher body weights, respectively (Table 4).
4. Discussion Based on egg production observations, there was no difference in the rate of egg production from the infection of the larvae into the body up to their growth and development to maturity and until producing eggs in non-PMSG and PMSG lambs. However, the number of eggs per gram of feces 4 weeks after infection was dramatically different; the 114
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contortus worms. This result confirms that the PMSG lambs had better resilience. This resilience capacity was also shown by the depression and reduction of egg production of the worms, showing that the immune response of the PMSG lambs was able to suppress and depress the fecundity of the infecting worms. The resilience was shown by the positive growth rate even though there was an infection of worms in their bodies. This result indicated that the bodies of the PMSG lambs were more tolerant to infection. In comparison, PMSG lambs maintained the positive body weight gain after infection, while non-PMSG lambs showed decreased body weights during the 3 months of observation after infection. In addition, non-infected PMSG lambs reached body weights of 28.33 kg, while non-infected non-PMSG lambs only reached 21.43 kg at the age of 8 months. In summary, the parameters of postnatal growth of infected and non-infected PMSG lambs clearly showed that PMSG lambs have better growth phenotypes and resilience to parasite infections. Our previous study showed that an improved uterine environment during pregnancy by improving the endogenous secretions of pregnancy hormones significantly improved prenatal growth and development as was indicated by the better birth weight and pre-weaning and post-weaning growth in sheep and swine (Manalu et al., 2000, 1998; Manalu and Sumaryadi, 1998) (Manalu et al., 2000; Manalu and Sumaryadi, 1998; Rayer et al., 2015a, b; Mege et al., 2006). In polytocous animals, the improved postnatal growth is also associated with increased survival as indicated by the lower morbidity and mortality (Rayer et al., 2015a, b; Mege et al., 2006). Observation in pigs showed that the improved growth phenotype of piglets produced by improvement of the uterine environment by improved endogenous secretion of pregnancy hormones also showed a better expression of growth hormone in the pituitary (Manampiring et al., 2016). The improved postnatal growth of the offspring produced by gonadotropin injection of the gilts prior to mating showed better postnatal growth and lower mortality and morbidity as well as a better response to vaccination (Montolalu et al., 2017). Since postnatal growth and health performance are determined by the uterine programming during prenatal growth and development during pregnancy, an improved uterine environment by increasing endogenous secretion of pregnancy hormones through the injection of the maternal animals with gonadotropin prior to mating significantly improved fetal prenatal growth and development that eventually improved pre-weaning and post-weaning growth and health performance. The improved uterine and placental structure and functions during pregnancy improved gene expression and protein abundance that eventually function as epigenetic signals affecting fetal growth and development as well as phenotype diversity, which will affect postnatal growth and physiological variability (Fowden et al., 2008; Fowden and Forhead, 2009a, b). The results of the present experiment strongly confirm that resilience of lambs to H. contortus can be produced with a simple technology by improving the uterine environment during pregnancy by injection of the ewes with gonadotropin prior to mating. In addition, the results obtained in the present experiment indicate that improved uterine environment during pregnancy by injection of maternal ewes prior to mating could produce a more resilience lambs that may help decontaminate pasture and reduce the number of anthelmintic treatment and use in lambs.
The PMSG lambs showed a higher immunological response as compared to non-PMSG lambs as indicated by the degree of the decrease in hemoglobin, hematocrit, and erythrocyte parameters; the decreases in the values were greater in the non-PMSG lambs compared to PMSG lambs. The greater decrease in red blood cells or erythrocyte parameters in non-PMSG lambs indicates that PMSG lambs have better recovery response compared to non-PMSG lambs. This indication can also be seen in the higher values of MCHC in PMSG lambs. The higher values of MCHC indicate the higher average of hemoglobin concentration in each red blood cell or erythrocyte so that the process of oxygen transport in the blood circulation is more optimal. The normal erythrocyte parameters in PMSG lambs, even though they faced a similar level of infection, supports the normal physiological status. The normal physiological condition supports normal metabolism and growth of the experimental lambs as indicated by the positive growth rate after infection. The decrease in erythrocyte and thrombocyte parameters in infected non-PMSG lambs as indications of lower resistance showed a decreased body weight during the first 3 months of infection. The higher number of thrombocytes in the PMSG lambs compared to control non-PMSG lambs indicates a more optimal response of stopping bleeding due to infection of H. contortus. With a higher thrombocyte parameter, the degree of blood loss can be reduced, and the infecting worms face greater difficulties in obtaining blood to meet their nutrient requirements. The higher number of eosinophil and lymphocyte percentages in the PMSG lambs as compared to non-PMSG lambs, even though there was no difference in leukocytes, may produce a superior immunity resistance of the PMSG lambs to infection of H. contortus. During parasite infections, it was stated that the response with higher eosinophil percentages was significant in the process of immune response (Behm and Ovington, 2000). Lymphocyte responses were improved if the responses of eosinophils and the other general immunity cells were optimal from the beginning of infection (Reinhardt et al., 2011). Therefore, the higher increases in lymphocyte and eosinophil percentages in infected PMSG lambs observed in this experiment indicate a better immune response as observed in higher resistance and resilience. In addition, the phenomena of higher lymphocyte concentrations with lower segmented neutrophils observed in PMSG lambs after infection indicate the faster immunity response of the PMSG lambs that are better than those of non-PMSG lambs. In the early response to infection, the concentrations of segment neutrophils will increase and will be higher compared to lymphocytes. In this experiment, observation of infection response was conducted for 3 months, which was the final stage of the sub-chronic condition and the beginning of the chronic infection condition. The optimum responses of the immune system to chronic infection are the increased or higher lymphocyte numbers and the decrease in the number of segmented neutrophils as was observed in the PMSG lambs in the present experiment. In addition, the lower number of monocytes in the blood circulation of the PMSG lambs indicates the higher number of monocytes that are actively converted into macrophages and further mobilized to enter the tissues to overcome or inhibit the worm infections that are in progress. Therefore, it is clear that the PMSG lambs have the more responsive immune system in response to the existence or presence of worm infections. These observations of the host response to infection, especially in PMSG lambs, strongly confirm that the PMSG lambs had higher fitness and resilience to infection. This result was indicated by the normal erythrocyte parameters, even though the experimental lambs were infected with the larvae of H. contortus. In addition, the higher immune response shown by the higher eosinophil number after infection also indicated a better immune system quality in the experimental PMSG lambs. The positive and higher growth rate of PMSG lambs after infection strongly confirm that PMSG lambs could resist the infection and increase their body weight despite an infection and infestation of H.
5. Conclusion The improvement of the uterine environment during pregnancy improves prenatal and postnatal growth of the offspring as well as resilience of the offspring to infection of H. contortus. The increased resilience of the offspring is reflected in the eosinophil and lymphocyte parameters. Improvement of the immunities of the offspring born to ewes having better uterine environments during pregnancy could depress the growth rate and fecundity of the infecting female H. contortus worms. The improvement of the uterine environment during pregnancy 115
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by improving endogenous secretions of pregnancy hormones during pregnancy can be used as a safe and inexpensive technique to produce superior offspring having better growth and immunity performance.
different litter sizes. Small Rumin. Res. 30, 163–169. Manalu, W., Sumaryadi, M.Y., Sudjatmogo, Satyaningtijas, A.S., 1998. Effect of superovulation on maternal serum progesterone concentration, uterine and fetal weights at weeks 7 and 15 of pregnancy in Javanese Thin-Tail ewes. Small Rumin. Res. 30, 171–176. Manalu, W., Sumaryadi, M.Y., Sudjatmogo, Satyaningtijas, A.S., 2000. The effect of superovulation of Javanese thin-tail ewes prior to mating on lamb birth weight and preweaning growth. Asian-Australas. J. Anim. Sci. 13, 292–299. Manampiring, N., Sumantri, C., Maheshwari, H., Manalu, W., 2016. Expression of growth hormone gene in the pituitary of piglets born to gilts Injected with pregnant mare serum gonadotropin and human chorionic gonadotropin prior to mating. Int. J. Sci. Basic Appl. Res. 30, 446–455. Marshall, K., Mugambi, J.M., Nagda, S., Sonstegard, T.S., Van Tassell, C.P., Baker, R.L., Gibson, J.P., 2013. Quantitative trait loci for resistance to Haemonchus contortus artificial challenge in Red Maasai and Dorper sheep of East Africa. Anim. Genetics 44, 285–295. Mege, R.A., Nasution, H.S., Kusumorini, N., Manalu, W., 2006. Effect of superovulation on piglet production (In Indonesian). Anim. Prod. 8, 8–15. Montolalu, F., Rita, D., Esfandiari, A., Manalu, W., 2017. The effect of vaccination on hematological profiles of piglets born to sows injected with gonadotropin prior to mating. Int. J. Sci. Basic Appl. Res. 33, 53–62. Rayer, D.J.J., Maheshwari, H., Muladno, Manalu, W., 2015a. Production of superior pigs by injecting the sows with gonadotropin prior to mating. Anim. Prod. 17, 8–15. Rayer, D.J.J., Muladno, Maheshwari, H., Manalu, W., 2015b. Improvement of growth phenotype of local piglet by gonadotrophin injection of sow prior to mating (In Indonesian). J. Veteriner 16, 599–605. Reinhardt, S., Scott, I., Simpson, H.V., 2011. Neutrophil and eosinophil chemotactic factors in the excretory/secretory products of sheep abomasal nematode parasites: NCF and ECF in abomasal nematodes. Parasitol. Res. 109, 627–635. Roberts, J.L., Swan, R.A., 1981. Quantitative studies of ovine haemonchosis. I. Relationship between faecal egg counts and total worm counts. Vet. Parasitol. 8, 165–171. Roberts, J.L., Swan, R.A., 1982. Quantitative studies of ovine haemonchosis. 2. Relationship between total worm counts of Haemonchus contortus, haemoglobin values and body weight. Vet. Parasitol. 9, 217–222. Roepstorff, A., Nansen, P., 1998. Epidemiology, Diagnosis and Control of Helminth Parasites of Swine. FAO Animal Health Manual, Rome, Italy, pp. 51–56. Rowe, A., McMaster, K., Emery, D., Sangster, N., 2008. Haemonchus contortus infection in sheep: parasite fecundity correlates with worm size and host lymphocyte counts. Vet. Parasitol. 153, 285–293. Sackett, D., Holmes, P., Abbott, K., Jephcott, S., Barber, M., 2006. Assessing the economic cost of endemic disease on the profitability of Australian beef cattle and sheep producers. Meat and Livestock Australia Report. Smith, W.D., Christie, M.G., 1979. Haemonchus contortus: some factors influencing the degree of resistance of sheep immunized with attenuated larvae. J. Comp. Pathol. 89, 141–150. Strain, S.A.J., Stear, M.J., 2001. The influence of protein supplementation on the immune response to Haemonchus contortus. Parasite Immunol. 23, 527–531. Vanimisetti, H.B., 2003. Genetics of Resistance to Haemoncus Contortus Infections in Sheep. Thesis. Virginia Tech. . Wanyangu, S.W., Mugambi, J.M., Bain, R.K., Duncan, J.L., Murray, M., Stear, M.J., 1997. Response to artificial and subsequent natural infection with Haemonchus contortus in Red Maasai and Dorper ewes. Vet. Parasitol. 69, 275–282.
Conflict of interest There is no conflict of interest related to the research and the publication. Acknowledgment This experiment was funded by the Ministry of Research, Technology, and Higher Education through the Program Magister Doktor Sarjana Unggul (PMDSU) in the year of 2016, with contract number: 180/SP2H/LT/DRM/III/2016. References Alba-Hurtado, F., Munoz-Guzman, M.A., 2013. Immune responses associated with resistance to haemonchosis in sheep. Biomed Res. Int. 2013, 1–11. Andriyanto, Amrozi, Rahminiwati, M., Boediono, A., Manalu, W., 2017. Optimum dose and time of pregnant mare serum gonadotropininjections in Kacang goats to increase endogenous secretion ofestrogen and progesterone without superovulation response. Small Rumin. Res. 149, 147–153. Arif, R., Andriyanto, Winarto, A., Boediono, A., Satrija, F., Manalu, W., 2018. Morphometric and Histometric Development of Uterus at the End of Embryonic Stage of Pregnancy in Does Injected With Gonadotropin Prior to Mating. Submitted to Small Ruminant Research. . Behm, C.A., Ovington, K.S., 2000. The role of eosinophils in parasitic helminth infections: insights from genetically modified mice. Parasitol. Today 16, 202–209. Fowden, A.L., Forhead, A.J., 2009a. Endocrine regulation of feto-placental growth. Horm. Res. 72, 257–265. Fowden, A.L., Forhead, A.J., 2009b. Hormones as epigenetic signals in developmental Programming. Exp. Physiol. 94, 607–625. Fowden, A.L., Forhead, A.J., Coan, P.M., Burton, G.J., 2008. The placenta and intrauterine programming. J. Neuroendocrinol. 20, 439–450. Ginting, S.P., Batubara, A., Romjali, E., Rangkuti, M., Subandriyo, 1999. Responses of two genotypes of lambs on the infection of Haemonchus contortus and the level of energy supplements. J. Imu Ternak dan Vet. 4, 20–27. Lacroux, C., Nguyen, T.H.C., Andreoletti, O., Prevot, F., Grisez, C., Bergeaud, J.P., Gruner, L., Brunel, J.C., Francois, D., Dorchies, P., Jacquiet, P., 2006. Haemonchus contortus (Nematoda: trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response. Vet. Res. 37, 607–622. Luffau, G., Khang, J., Bouix, J., Nguyen, T., Cullen, P., Ricordeau, G., Carrat, C., Eychenne, F., 1990. Resistance to experimental infections with Haemonchus contortus in Romanov sheep. Genet. Sel. Evol. 22, 205–229. Manalu, W., Sumaryadi, M.Y., 1998. Maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition in Javanese Thin-Tail ewes with
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Production of lambs’ resilience to Haemonchus contortus Ridi Arif
a,b
b
c
c
, Fadjar Satrija , Adi Winarto , Arief Boediono , Wasmen Manalu
c,⁎
T
a
Graduate Student of Animal Physiology and Pharmacology, Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia Department of Animal Diseases and Veterinary Health, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia c Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Jl Agatis, Kampus IPB Darmaga, Bogor, 16680, West Java, Indonesia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lamb resilience Haemonchus contortus Intrauterine programming Gonadotropin
Haemonchus (H.) contortus infection in sheep is a major health problem in tropical and sub-tropical regions that cause great economic losses. Our previous studies have shown that improved uterine environments during pregnancy can improve postnatal growth and health performance of the offspring, indicated by lower mortality and morbidity. In the present experiment, we evaluated the resistance and resilience to H. contortus of lambs born to ewes injected with pregnant mare’s serum gonadotropin (PMSG) prior to mating. Improvement of the uterine environment was conducted by increasing endogenous secretion of estrogen and progesterone as pregnancy hormones during pregnancy by injecting the ewes with PMSG prior to mating. A total of 16 lambs, regardless of sex, at the age of 5 months were assigned into a 2 × 2 factorial experiment with 4 replications. The first factor was PMSG injection, consisting of two levels, i.e., lambs born to ewes without PMSG injection (NonPMSG lambs) and those born to PMSG-injected ewes (PMSG lambs). The second factor was the infection of lambs with H. contortus at the age of 5 months, consisting of two levels, i.e., lambs without infection (Non-infected lambs) and lambs infected with H. contortus (Infected lambs). Non-infected lambs were administered with distilled water in a capsule without infective larvae. Infected lambs were individually infected with a single dose containing 1200 L3 of H. contortus. Compared to non-PMSG lambs, PMSG lambs tended to have better prenatal growth indicated by greater birth weights (P = 0.06). The improved prenatal growth during pregnancy improved the postnatal growth and health performance of the lambs. Three months after infection of H. contortus, non-PMSG lambs and PMSG lambs had similar worm counts. However, the PMSG lambs showed significantly higher resilience to H. contortus as indicated by the lower fecal egg counts 6–10 weeks after infection. The higher resilience of the PMSG lambs was shown by the positive growth rate during infection, while non-PMSG lambs had a negative growth rate after infection. Prior to infection, PMSG lambs showed a higher segmented neutrophil percentage with lower lymphocyte numbers. Three months after infection, PMSG lambs had significantly higher lymphocyte and thrombocyte numbers as well as mean corpuscular hemoglobin concentration (MCHC) with lower neutrophil and monocyte numbers. The conclusion of this study is that the improvement of the uterine environment during pregnancy could be used to produce superior offspring with high resilience to the infection of H. contortus.
1. Introduction Parasite infestation by nematode in the intestine is a common problem in the animal husbandry industry. The infection of this parasite increases the operation costs and causes economic losses (Sackett et al., 2006). In addition, the problem is worse with the report that some intestinal parasites have increased resistance due to inappropriate and uncontrolled use of anthelmintics (Luffau et al., 1990). One of the main
⁎
gastrointestinal parasites in sheep is the nematode worm of Haemonchus (H.) contortus. H. contortus infection causes decreased body weight and anemia (Strain and Stear, 2001). In the case of chronic infection, the decreases in body weight and hematocrit levels are not normally found as compared to the non-infected animals. In hyper-acute cases, the infected animals can die quickly due to the serious bleeding in the gastrointestinal tract (Roberts and Swan, 1982). Several experiments have been conducted to map factors increasing
Corresponding author. E-mail address: [email protected] (W. Manalu).
https://doi.org/10.1016/j.smallrumres.2018.08.016 Received 7 February 2018; Received in revised form 20 June 2018; Accepted 20 August 2018 Available online 23 August 2018 0921-4488/ © 2018 Elsevier B.V. All rights reserved.
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contortus (infected lambs). The lambs in the infected lambs group were individually infected with a single dose containing 1200 L3 of H. contortus (modified from Ginting et al., 1999), while the non-infected group was administered with distilled water in a capsule without H. contortus. The experiment was conducted according to the National Institute of Health’s guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978).
the resistances of animals to H. contortus. One of the factors that has become a popular topic in veterinary parasitology is the genes that control the resistance phenotype (Alba-Hurtado and Munoz-Guzman, 2013). However, it is known that the genetically resistance mother does not always produce resistant offspring because the inheritance of the resistance phenotype to the offspring is not absolute (Marshall et al., 2013). Therefore, crossing with a strict selection can be used to produce offspring having high resistance. However, the method takes longer and requires strict record keeping (Wanyangu et al., 1997). Therefore, a more efficient new approach or method is required to optimize and increase the expression of the resistance genotype in offspring. Based on the understanding that the resistance phenotype is a form of united and coordinated function contributed by some bodily functions, to optimize and activate the resistance phenotypes, the improvement of all body systems is required. One approach is to optimize the development of the organ systems during the growth and development of the embryo and fetus during pregnancy. Improvement of growth and development of the embryo and fetus during pregnancy can be achieved by increasing the signals and factors controlling the growth and development of the uterine and placenta (Fowden et al., 2008; Fowden and Forhead, 2009a) by optimizing the availabilities of pregnancy hormones during pregnancy. Improved uterine and placental structure and functions during pregnancy improve gene expression and protein abundance that eventually function as epigenetic signals that affect fetal growth and development as well as phenotype diversity, which affects postnatal growth and physiological variability (Fowden and Forhead, 2009b). Maternal serum progesterone concentrations, as the main pregnancy hormones, can be increased by gonadotropin injection of the mothers prior to mating that eventually improves prenatal and postnatal growth in sheep and swine (Manalu et al., 2000; Manalu and Sumaryadi, 1998; Rayer et al., 2015a, b; Mege et al., 2006). The improved postnatal growth also associates with the increased survival as indicated by the lower morbidity and mortality (Rayer et al., 2015a, b; Mege et al., 2006). Based on these phenomena, the present experiment was designed to evaluate the resistance and resilience to H. contortus of lambs born to ewes injected with pregnant mare's serum gonadotropin (PMSG) prior to mating. PMSG injection prior to mating increases endogenous secretion of pregnancy hormones that improve the uterine environment during pregnancy. Our previous studies have shown that PMSG-injected does prior to mating had greater uterus morphometric and histometric parameters that correlated well with the fetal weight at the end of embryonic stage of pregnancy (Arif et al., 2018). This is the first report on the improvement of lambs’ resistance and resilience to H. contortus by injecting the mother with PMSG prior to mating to improve the growth and development of the uterus and placenta that will support the prenatal growth of the offspring during pregnancy.
2.2. Experimental animals To produce lambs used in the experiment, all maternal ewes were injected with prostaglandin F2 alpha (PGF2α) two times with an 11-day interval to synchronize the estrus cycle prior to mating. The PMSGinjected ewes were injected with PMSG at a dose of 7.5 IU/kg body weight (BW) at the same time as the second PGF2α injection (Andriyanto et al., 2017). The non-PMSG-injected ewes were injected with 0.7% NaCl solution. At the estrus period, all experimental ewes were mated naturally by mixing with selected male sheep for 1 week. The experimental ewes were maintained until parturition. At parturition, the birth weights of the lambs were measured. At weaning time at the age of 20 weeks postpartum, the lambs were selected for infection of infective H. contortus larvae. Prior to infection, the experimental lambs were raised intensively in cages with the maternal ewes. During the treatment, the experimental lambs were maintained intensively in cages. 2.3. Production of infective H. Contortus larvae, artificial infection, and collecting samples Two donor sheep were prepared for production of infective H. contortus larvae. The donor sheep were administered anthelmintics for 3 consecutive days. The number of eggs in each gram of feces was measured, and the sheep were free of worm infection. The donor sheep were acclimatized for 1 week. The donor sheep were fed with grass and nongrass forage feed free of worm eggs and larvae. The mature female H. contortus worms were collected from the abomasum of sheep from the local slaughterhouse. The mature worms were collected, grinded to obtain eggs, and then cultured and nourished. After 1 week of culturing, the collection of L3 larvae was conducted. The collected L3 larvae were put into a soft capsule for further use to infect the donor experimental sheep. Two weeks after infection, the number of eggs per gram of feces was counted to ensure that the donor sheep were infected with H. contortus, and the eggs were found in the feces. The feces of the donor sheep was cultured for further collection of L3. The dose of L3 infection was estimated by calculating the concentrations, i.e., the number of L3 larvae per milliliter of distilled water. The number of infected L3 larvae was 1200 L3 larvae per experimental lamb (modified from Ginting et al. (1999).
2. Materials and methods 2.4. Infection of experimental lambs 2.1. Experimental design The experimental lambs were infected with infective larvae packed in a capsule directly into the stomach using a stomach tube. The uninfected lambs were administered with distilled water in a capsule without infective larvae. After infection, the fecal samples were collected weekly up to 13 weeks post-infection to measure the number of worm eggs in the feces or fecal egg count (FEC). Thirteen weeks after infection, the fecal samples were collected for 24 h to measure the total worm eggs to calculate female worm fecundity. At the end of experiment (13 weeks post infection), the blood samples were also collected to measure blood cell parameters (erythrocyte, leukocytes, and thrombocytes). After collecting blood samples, the infected lambs (nonPMSG lambs and PMSG lambs) were sacrificed to obtain the abomasum for measurement of the number of male and female worms, total number of male and female worms, and the length of male and female worms in the mucosa of the abomasum. Since the non-infected lambs
An experiment was designed in a completely randomized fashion with 2 × 2 factorial arrangement, and each experimental unit used 4 lambs. The experiment used 8 lambs born to PMSG-injected ewes (PMSG lambs) and 8 lambs born to non-PMSG injected ewes (nonPMSG lambs). The age of the experimental lambs at the beginning of experiment was 5 months, and there was no sexual grouping of the experimental lambs. The first factor was the PMSG injection, consisting of two levels, i.e., lambs born to ewes without PMSG injection prior to mating (non-PMSG lambs) and lambs born to PMSG-injected ewes prior to mating to improve endogenous secretions of pregnancy hormones to optimize the uterine environment during pregnancy (PMSG lambs). The second factor was the infection of the experimental lambs with H. contortus, consisting of two levels, i.e., lambs without infection H. contortus as a control (non-infected lambs) and lambs infected with H. 111
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2.6. Data analysis
Table 1 The number of male worms, female worms, total number of male and female worms, the length of male worms, the length of female worms, the number of worm eggs in 24 h, and the fecundity of female worms in the abomasum of nonPMSG lambs and PMSG lambs 3 months after infection with Haemonchus contortus. Variables
Non-PMSG lambs
The number of male worms (worms) The number of female worms (worms) Total number of worms (worms) The length of male worms (cm) The length of female worms (cm) The number of eggs in 24 hours (eggs) The fecundity of female worms (eggs/worm)
14.20 ± 3.30 3.80 ± 1.41
The collected data were analyzed with analysis of variance (SPSS 16). The significant effects of main factor (PMSG injection of the ewes and infection of the experimental lambs) and their interactions were tested. The significant differences between experimental units were further tested by the Duncan ranges test (SPSS 16) using α = 0.05 as a level of significance.
PMSG lambs
a
14.25 ± 4.57
a
4.00 ± 2.16
a
a
3. Results 18.00 ± 6.36 a 1.39 ± 0.08 a 1.80 ± 0.31 a 2143.13 ± 424.26 563.98 ± 298.56 a
a
18.25 ± 5.91 a 1.42 ± 0.05 a 1.34 ± 0.86 a 1885.00 ± 523.26
During the 4-week period after infection, there was no FEC found in the feces of infected lambs (Fig. 1). The number of FEC increased with time 5 weeks after infection and reached the peak level 9 weeks after infection (P < 0.05). Five weeks after infection, PMSG lambs had numerically lower FEC compared to non-PMSG lambs (P = 0.282). PMSG lambs had consistently lower FEC compared to non-PMSG lambs 6–9 weeks after infection (P < 0.05) (Fig. 1). During the period of 6, 7, 8, and 9 weeks after infection, PMSG lambs consistently had lower FEC by 51.85, 57.58, 51.61, and 38.89%, respectively, compared to non-PMSG lambs (P < 0.05). Then, 10–13 weeks after infection, the FEC of nonPMSG lambs decreased slightly with the same concentrations with those of PMSG lambs (P > 0.05). Although there was a significant decrease in the FEC of PMSG lambs 6–9 weeks after infection, the number and morphometric measurement of adult worms in the abomasum of the lambs 13 weeks after infection did not show significant differences (Table 1). The number of male worms, female worms, and the total number of worms, the lengths of male and female worms, the number of eggs in 24 h, and the fecundity of female worms in the abomasum of non-PMSG lambs and PMSG lambs 13 weeks after infection did not show significant differences (Table 1) (P > 0.05). However, there was still a tendency of numerically shorter length of female worms, lower number of eggs in 24 h, and lower fecundity and fertility of female worms in the PMSG lambs compared to non-PMSG lambs (Table 1). Prior to infection of L3 H. contortus at the weaning period, the blood parameters of hemoglobin, leukocytes, thrombocytes, hematocrit, erythrocytes, MCV, MCH, MCHC, eosinophils, band neutrophils, segment neutrophils, lymphocytes, and monocytes in all groups of experimental lambs were similar (P > 0.05) (Table 2). The similar erythrocyte parameters in PMSG and non-PMSG lambs indicated that both groups of experimental lambs had similar normal physiological conditions at the weaning period before infection. The basophils were not detected in non-PMSG and PMSG lambs. Even though there was no statistical significance in leukocyte parameters in all groups of experimental lambs
a
471.25 ± 120.29 a
a,b
Different superscripts in the same row indicate a significant difference (p < 0.05).
did not have infection, the non-infected lambs were not sacrificed. Therefore the data in Table 1 and Fig. 1 only reflects PMSG and nonPMSG lambs infected with H. contortus. Body weights of the experimental lambs were measured at lambing, 4 months postpartum (1 month before infection), 7 months postpartum (2 months after infection), and 8 months postpartum (3 months after infection).
2.5. Parameters measured The parameters measured in this experiment were the blood parameters (hemoglobin, leukocytes, thrombocytes, hematocrit, erythrocytes, average red blood cell size (MCV), hemoglobin amount per red blood cell (MCH), mean corpuscular hemoglobin concentration MCHC, basophils, eosinophils, band neutrophils, segment neutrophils, lymphocytes, and monocytes) of the experimental lambs before treatment (at the age of 5 months) and at the end of experiment (13 weeks post infection). Hematology parameters were measured by a hematology analyzer (Sysmex XP-300®). FEC was measured by the McMaster method (Roepstorff and Nansen 1998) in Laboratory of Helminthology of the Faculty of Veterinary Medicine of Bogor Agricultural University. The total number of worms (male and female) was counted manually. The length of the worms was measured using a caliper. Body weights of the experimental lambs were measured using a digital scale.
Fig. 1. Fecal egg count (FEC) of non-PMSG lambs (■) and PMSG lambs (♦)3 months after infection of Haemonchus contortus. a,b Different superscripts in the same weeks after infection indicate a significant difference (p < 0.05). 112
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Table 2 Red blood cell, thrombocyte, and leukocyte profiles of non-PMSG lambs and PMSG lambs before infection with Haemonchus contortus. Variables
Hemoglobin (g/dL) Leukocytes (103xμL) Thrombocytes (103xμL) Hematocrit (%) Erythrocytes (106xμL) MCV (fl) MCH (fg) MCHC (%) Basophils (%) Eosinophils (%) Band Neutrophils (%) Segmented Neutrophils (%) Lymphocytes (%) Monocytes (%)
Non-PMSG lambs
PMSG lambs
Level of significance (p value)
Non-Infected
Infected
Non-Infected
Infected
PMSG
Infection
Interaction
12.53 ± 0.26 9.55 ± 1.00 309.75 ± 62.30 37.50 ± 0.58 4.15 ± 0.06 90.00 ± 0.00 30.00 ± 0.00 33.25 ± 0.50 0.00 ± 0.00 0.50 ± 0.58 1.50 ± 1.00 68.75 ± 2.75 24.50 ± 2.08 4.75 ± 1.50
13.14 ± 0.54 11.78 ± 2.90 379.80 ± 70.67 40.20 ± 1.50 4.38 ± 0.22 89.80 ± 0.82 30.00 ± 0.00 33.20 ± 0.58 0.00 ± 0.00 0.80 ± 0.50 1.40 ± 1.00 69.60 ± 1.83 22.40 ± 4.19 5.80 ± 1.73
13.43 ± 1.56 12.38 ± 4.95 354.25 ± 26.89 40.25 ± 4.76 4.43 ± 0.50 90.00 ± 0.84 30.00 ± 0.00 33.50 ± 0.45 0.00 ± 0.00 0.75 ± 0.45 1.50 ± 0.89 77.00 ± 8.35 16.75 ± 6.11 5.50 ± 1.79
13.30 ± 1.24 12.85 ± 3.94 377.50 ± 37.50 39.75 ± 3.50 4.43 ± 0.40 89.75 ± 0.50 30.00 ± 0.00 33.50 ± 0.58 0.00 ± 0.00 0.25 ± 0.50 0.25 ± 0.50 75.25 ± 6.08 19.25 ± 5.25 5.00 ± 1.41
0.337 0.297 0.411 0.477 0.371 0.939 – 0.300 – 0.552 0.200 0.027 0.036 0.975
0.652 0.461 0.083 0.496 0.517 0.492 – 0.923 – 0.690 0.137 0.874 0.933 0.735
0.498 0.630 0.363 0.327 0.517 0.939 – 0.923 – 0.127 0.200 0.648 0.342 0.347
affected the segmented neutrophil percentages (P < 0.01). However, there was a significant interaction effect of infection and PMSG injection on segmented neutrophil percentages (P < 0.01) (Table 3). In PMSG lambs, infection significantly decreased segmented neutrophil percentages (P < 0.05). However, in non-PMSG lambs, infection did not affect the percentage of segmented neutrophils (P > 0.05). The lymphocyte percentage profiles of experimental lambs was similar to that of segmented neutrophils, in which there was a significant effect of infection (P < 0.01) and interaction between the PMSG injection and infection (P < 0.01) without a significant effect of PMSG injection (P > 0.05) (Table 3). In the non-PMSG lambs, infection did not change the lymphocyte percentage (P > 0.05). However, in PMSG lambs, infection increased the lymphocyte percentage (P < 0.05), while it decreased the segmented neutrophil percentage (P < 0.05). Different from lymphocyte profiles, and similar to the profiles of hemoglobin, hematocrit, and erythrocytes, infection of non-PMSG and PMSG lambs decreased the monocyte percentage observed 13 weeks after infection (Table 3). PMSG injection did not affect monocyte concentrations, and there was no interaction of infection and PMSG injection on monocyte percentages (P > 0.05) (Table 3). Regardless of PMSG injection, infection tended to decrease thrombocyte concentrations observed 13 weeks after infection (P = 0.054). Regardless of infection, PMSG and non-PMSG lambs had similar thrombocyte concentrations (P > 0.05) (Table 3). There was no interaction of infection and PMSG injection on thrombocyte
prior to infection, the PMSG lambs showed a tendency of a higher number of leukocytes. However, prior to infection, the percentage of lymphocytes in the non-PMSG lambs were higher compared to those of PMSG lambs (P < 0.05), and the percentage of segmented neutrophils in PMSG lambs were higher compared to non-PMSG lambs (P < 0.05). All these blood parameters were similar in infected and non-infected lambs. However, 3 months after infection, changes in blood parameters were observed (Table 3). In general, all groups of experimental lambs had similar leukocyte, MCV, MCH, basophil, eosinophil, and band neutrophil parameters (P > 0.05). No basophils were observed in any experimental lambs in this experiment. The percentages of MCHC in the experimental lambs were higher compared to non-PMSG lambs (P < 0.05) (Table 3). Infection significantly decreased hemoglobin concentrations, hematocrit percentages, and erythrocyte concentrations regardless of PMSG injection (P < 0.01). However, regardless of infection, PMSG lambs had similar hemoglobin concentrations, hematocrit percentages, and erythrocyte concentrations (P > 0.05) (Table 3). There was no interaction between infection and PMSG injection on hemoglobin concentrations, hematocrit percentage, and erythrocyte concentrations of the experimental lambs (P > 0.05). The profile of segmented neutrophils was different from that of band neutrophils. PMSG injection and infection did not affect band neutrophil percentages (P > 0.05). In contrast, infection significantly
Table 3 Red blood cell, thrombocyte, and leukocyte profiles of non-PMSG lambs and PMSG lambs 3 months after infection with Haemonchus contortus. Variables
Hemoglobin (g/dL) Leukocytes (103xμL) Thrombocytes (103xμL) Hematocrit (%) Erythrocytes (106xμL) MCV (fl) MCH (fg) MCHC (%) Basophils (%) Eosinophils (%) Band Neutrophils (%) Segmented Neutrophils (%) Lymphocytes (%) Monocytes (%) a,b
Non-PMSG lambs
PMSG lambs
Level of significance (p value)
Non-Infected
Infected
Non-Infected
Infected
PMSG
Infection
Interaction
13.14 ± 1.56 11.78 ± 4.95 379.80 ± 26.89 40.20 ± 4.76 4.38 ± 0.50 89.80 ± 0.84 30.00 ± 0.00 33.20 ± 0.45 0.00 ± 0.00 0.80 ± 0.45 1.40 ± 0.89 69.60 ± 8.35 b 22.40 ± 6.11 b 5.80 ± 1.79
11.56 ± 0.86 11.84 ± 1.39 269.80 ± 100.08 34.60 ± 2.30 3.84 ± 0.27 90.00 ± 0.71 30.00 ± 0.00 33.60 ± 0.55 0.00 ± 0.00 0.40 ± 0.55 1.20 ± 0.84 70.40 ± 2.41 b 24.40 ± 2.19 b 3.60 ± 0.89
14.07 ± 0.53 14.87 ± 2.90 378.00 ± 38.05 42.00 ± 1.41 4.67 ± 0.17 89.75 ± 0.96 30.00 ± 0.00 33.75 ± 0.50 0.00 ± 0.00 0.50 ± 0.58 1.00 ± 1.15 78.75 ± 2.50 a 14.75 ± 3.86 c 5.00 ± 1.83
11.92 ± 0.44 10.55 ± 3.16 361.25 ± 55.05 36.00 ± 1.15 3.97 ± 0.15 90.00 ± 0.00 30.00 ± 0.00 34.00 ± 0.00 0.00 ± 0.00 1.25 ± 0.95 2.25 ± 1.71 59.50 ± 6.35 c 34.50 ± 4.04a 2.50 ± 0.58
0.195 0.584 0.159 0.272 0.179 0.944 – 0.040 – 0.381 0.563 0.748 0.560 0.171
0.002 0.206 0.054 0.001 0.001 0.529 – 0.144 – 0.574 0.355 0.004 0.000 0.003
0.560 0.194 0.144 0.888 0.607 0.944 – 0.727 – 0.079 0.208 0.002 0.001 0.823
Different superscripts in the same row indicate a significant difference (p < 0.05). 113
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Table 4 Body weights of non-PMSG lambs (control) and PMSG lambs at lambing (birth weight), 1 month before infection (at the age of 4 months), and 2 and 3 months after infection with Haemonchus contortus. Time
Non-PMSG lambs
Non-infected Birth weight (kg) Body weight (kg) 1 month preinfection 2 months postinfection 3 months postinfection a,b
1.15 ± 0.11 15.42 ± 4.13
PMSG lambs
Infected
b
19.90 ± 4.41ab 21.43 ± 5.19ab
1.2 ± 0.05 16.16 ± 1.23
b
15.74 ± 1.98b 14.9 ± 1.77b
Level of significance (p value)
Non-infected
Infected
2.67 ± 0.26 20.70 ± 2.08a
2.19 ± 0.96 17.55 ± 2.79
24.96 ± 3.52a 28.33 ± 5.90a
19.42 ± 4.08ab 22.15 ± 5.25ab
ab
PMSG
Infection
Interaction
0.066 0.071
0.619 0.112
0.691 0.254
0.032* 0.024*
0.043* 0.032*
0.942 0.991
Different superscripts in the same row indicate a significant difference (p < 0.05).
PMSG lambs had only 230 eggs/g of feces while the non-PMSG lambs had 620 eggs/g of feces. The FEC has higher correlation with total worm counts (Roberts and Swan, 1981). The pattern of increase in the number of worm eggs per gram of feces was slightly slower in the PMSG lambs compared to non-PMSG lambs even though it further decreased and met the same level 12 weeks after infection. Previous studies have reported that the decrease in egg numbers per gram of feces generally occurred 14 weeks after infection and was followed by the constant values of eggs per gram of feces (Wanyangu et al., 1997). Since the worm number was not different, the lower number of eggs per gram of feces in the PMSG lambs indicates that the PMSG lambs have capacities to inhibit or depress the re-productivity and productivity of H. contortus worms. This assumption was based the results of blood parameters (Table 2). The improved quality of the immune system in the PMSG lambs that was shown by the higher number of eosinophils eventually decreased egg production by H. contortus. Observation of the number of worms showed that there was no difference between non-PMSG lambs and PMSG lambs. Even though the number of adult female worms was similar, the productivity of the adult female worms in the PMSG lambs was decreased. The decreased fecundity in adult female worms is highly correlated with the length of the adult female worms (Rowe et al., 2008). The numerically shorter length of adult female worms obtained from the PMSG lambs could be related to the decreased fecundity of female H. contortus worms. The higher number of lymphocytes observed in the PMSG lambs 3 months after infection may have contributed to the inhibition of worm fecundity. The higher number of lymphocytes, the increased activity of Thelper 2 (Th2) cells, the higher number of eosinophils in the abomasum, the increased number of mast cells, the increased concentrations of interleukin (IL)-4, IL-13, immunoglobulin (Ig)G, and IgA were shown to be good resistance features (Smith and Christie, 1979; Strain and Stear, 2001; Lacroux et al., 2006). The treatment could improve the immune response against gastrointestinal parasites that could influence the resilience of the lambs. The conditions of experimental lambs prior to infection were similar except that PMSG lambs had higher segmented neutrophil and lower lymphocyte percentages. These phenomena indicate the potency of higher immune system in PMSG lambs. Therefore, PMSG lambs had a higher ratio of segmented neutrophils and lymphocytes, indicating the physiological immune systems of PMSG lambs were more optimum. In general, the blood cell parameters observed in the experimental lambs prior to infection at the weaning period were similar to those reported in the region of Southeast Asia (Ginting et al., 1999). The similar decrease in the erythrocyte and leukocyte parameters of both groups of experimental lambs after infection strongly confirmed that infection was successful and produced pathological conditions (Rowe et al., 2008) in the experimental lambs. In addition, the average time required of 4 weeks from the artificial infection until the presence of worm eggs in the feces is similar to that reported previously (Vanimisetti, 2003).
concentrations (P > 0.05), indicating that PMSG lambs showed lower decreases in thrombocyte concentrations in response to H. contortus infection. The most important response to be highlighted related to the infection of H. contortus worms is the decrease in body weight of the infected lambs (Table 4). At birth, PMSG lambs had numerically higher birth weight (P = 0.066), and lambs in the group of infection had similar birth weights. One month prior to infection (at the age of 4 months), the same pattern of body weight in the experimental lambs was similar to the birth weight, in which PMSG lambs tended to have higher body weights compared to non-PMSG lambs (P = 0.071). Infected lambs had similar body weights 1 month before infection (P > 0.05). However, 2–3 months after infection, PMSG injection and infection significantly affected body weights of the experimental lambs. There was no interaction of PMSG injection and infection on the body weight of experimental lambs. PMSG lambs had significantly higher body weights compared to non-PMSG lambs during the period of 2 and 3 months after infection. Two months post-infection, infected PMSG lambs had 23.38% higher body weights compared to infected non-PMSG lambs, indicating that PMSG lambs had better growth response to infection compared to non-PMSG lambs. In the same period, non-infected PMSG lambs had a 25.43% higher body weight compared non-infected non-PMSG lambs, indicating the PMSG lambs had a significantly (P < 0.05) higher growth rate during the post-weaning period. Three months post-infection, infected PMSG lambs had a 48.66% higher body weight compared to infected non-PMSG lambs. In the same period, non-infected PMSG lambs had a 32.20% higher body weight compared to non-infected non-PMSG lambs, indicating that the PMSG lambs had better growth rates during the post-weaning period (P < 0.05). Regardless of PMSG injection, infection decreased body weight by 21.81% in PMSG lambs and 30.47% in non-PMSG lambs. This experiment showed that the improved resilience of PMSG lambs as indicated by the blood parameters responses could maintain the normal body functions as indicated by the increased body weight by 26.21% 3 months after infection compared to that 1 month prior to infection. In contrast, non-PMSG lambs had a negative growth rate (decreased body weight by 7.80%) 3 months after infection compared to that 1 month prior to infection. As a comparison, during the same period of measurements, non-infected PMSG lambs and non-infected non-PMSG lambs had 36.86% and 38.98% higher body weights, respectively (Table 4).
4. Discussion Based on egg production observations, there was no difference in the rate of egg production from the infection of the larvae into the body up to their growth and development to maturity and until producing eggs in non-PMSG and PMSG lambs. However, the number of eggs per gram of feces 4 weeks after infection was dramatically different; the 114
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contortus worms. This result confirms that the PMSG lambs had better resilience. This resilience capacity was also shown by the depression and reduction of egg production of the worms, showing that the immune response of the PMSG lambs was able to suppress and depress the fecundity of the infecting worms. The resilience was shown by the positive growth rate even though there was an infection of worms in their bodies. This result indicated that the bodies of the PMSG lambs were more tolerant to infection. In comparison, PMSG lambs maintained the positive body weight gain after infection, while non-PMSG lambs showed decreased body weights during the 3 months of observation after infection. In addition, non-infected PMSG lambs reached body weights of 28.33 kg, while non-infected non-PMSG lambs only reached 21.43 kg at the age of 8 months. In summary, the parameters of postnatal growth of infected and non-infected PMSG lambs clearly showed that PMSG lambs have better growth phenotypes and resilience to parasite infections. Our previous study showed that an improved uterine environment during pregnancy by improving the endogenous secretions of pregnancy hormones significantly improved prenatal growth and development as was indicated by the better birth weight and pre-weaning and post-weaning growth in sheep and swine (Manalu et al., 2000, 1998; Manalu and Sumaryadi, 1998) (Manalu et al., 2000; Manalu and Sumaryadi, 1998; Rayer et al., 2015a, b; Mege et al., 2006). In polytocous animals, the improved postnatal growth is also associated with increased survival as indicated by the lower morbidity and mortality (Rayer et al., 2015a, b; Mege et al., 2006). Observation in pigs showed that the improved growth phenotype of piglets produced by improvement of the uterine environment by improved endogenous secretion of pregnancy hormones also showed a better expression of growth hormone in the pituitary (Manampiring et al., 2016). The improved postnatal growth of the offspring produced by gonadotropin injection of the gilts prior to mating showed better postnatal growth and lower mortality and morbidity as well as a better response to vaccination (Montolalu et al., 2017). Since postnatal growth and health performance are determined by the uterine programming during prenatal growth and development during pregnancy, an improved uterine environment by increasing endogenous secretion of pregnancy hormones through the injection of the maternal animals with gonadotropin prior to mating significantly improved fetal prenatal growth and development that eventually improved pre-weaning and post-weaning growth and health performance. The improved uterine and placental structure and functions during pregnancy improved gene expression and protein abundance that eventually function as epigenetic signals affecting fetal growth and development as well as phenotype diversity, which will affect postnatal growth and physiological variability (Fowden et al., 2008; Fowden and Forhead, 2009a, b). The results of the present experiment strongly confirm that resilience of lambs to H. contortus can be produced with a simple technology by improving the uterine environment during pregnancy by injection of the ewes with gonadotropin prior to mating. In addition, the results obtained in the present experiment indicate that improved uterine environment during pregnancy by injection of maternal ewes prior to mating could produce a more resilience lambs that may help decontaminate pasture and reduce the number of anthelmintic treatment and use in lambs.
The PMSG lambs showed a higher immunological response as compared to non-PMSG lambs as indicated by the degree of the decrease in hemoglobin, hematocrit, and erythrocyte parameters; the decreases in the values were greater in the non-PMSG lambs compared to PMSG lambs. The greater decrease in red blood cells or erythrocyte parameters in non-PMSG lambs indicates that PMSG lambs have better recovery response compared to non-PMSG lambs. This indication can also be seen in the higher values of MCHC in PMSG lambs. The higher values of MCHC indicate the higher average of hemoglobin concentration in each red blood cell or erythrocyte so that the process of oxygen transport in the blood circulation is more optimal. The normal erythrocyte parameters in PMSG lambs, even though they faced a similar level of infection, supports the normal physiological status. The normal physiological condition supports normal metabolism and growth of the experimental lambs as indicated by the positive growth rate after infection. The decrease in erythrocyte and thrombocyte parameters in infected non-PMSG lambs as indications of lower resistance showed a decreased body weight during the first 3 months of infection. The higher number of thrombocytes in the PMSG lambs compared to control non-PMSG lambs indicates a more optimal response of stopping bleeding due to infection of H. contortus. With a higher thrombocyte parameter, the degree of blood loss can be reduced, and the infecting worms face greater difficulties in obtaining blood to meet their nutrient requirements. The higher number of eosinophil and lymphocyte percentages in the PMSG lambs as compared to non-PMSG lambs, even though there was no difference in leukocytes, may produce a superior immunity resistance of the PMSG lambs to infection of H. contortus. During parasite infections, it was stated that the response with higher eosinophil percentages was significant in the process of immune response (Behm and Ovington, 2000). Lymphocyte responses were improved if the responses of eosinophils and the other general immunity cells were optimal from the beginning of infection (Reinhardt et al., 2011). Therefore, the higher increases in lymphocyte and eosinophil percentages in infected PMSG lambs observed in this experiment indicate a better immune response as observed in higher resistance and resilience. In addition, the phenomena of higher lymphocyte concentrations with lower segmented neutrophils observed in PMSG lambs after infection indicate the faster immunity response of the PMSG lambs that are better than those of non-PMSG lambs. In the early response to infection, the concentrations of segment neutrophils will increase and will be higher compared to lymphocytes. In this experiment, observation of infection response was conducted for 3 months, which was the final stage of the sub-chronic condition and the beginning of the chronic infection condition. The optimum responses of the immune system to chronic infection are the increased or higher lymphocyte numbers and the decrease in the number of segmented neutrophils as was observed in the PMSG lambs in the present experiment. In addition, the lower number of monocytes in the blood circulation of the PMSG lambs indicates the higher number of monocytes that are actively converted into macrophages and further mobilized to enter the tissues to overcome or inhibit the worm infections that are in progress. Therefore, it is clear that the PMSG lambs have the more responsive immune system in response to the existence or presence of worm infections. These observations of the host response to infection, especially in PMSG lambs, strongly confirm that the PMSG lambs had higher fitness and resilience to infection. This result was indicated by the normal erythrocyte parameters, even though the experimental lambs were infected with the larvae of H. contortus. In addition, the higher immune response shown by the higher eosinophil number after infection also indicated a better immune system quality in the experimental PMSG lambs. The positive and higher growth rate of PMSG lambs after infection strongly confirm that PMSG lambs could resist the infection and increase their body weight despite an infection and infestation of H.
5. Conclusion The improvement of the uterine environment during pregnancy improves prenatal and postnatal growth of the offspring as well as resilience of the offspring to infection of H. contortus. The increased resilience of the offspring is reflected in the eosinophil and lymphocyte parameters. Improvement of the immunities of the offspring born to ewes having better uterine environments during pregnancy could depress the growth rate and fecundity of the infecting female H. contortus worms. The improvement of the uterine environment during pregnancy 115
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by improving endogenous secretions of pregnancy hormones during pregnancy can be used as a safe and inexpensive technique to produce superior offspring having better growth and immunity performance.
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Conflict of interest There is no conflict of interest related to the research and the publication. Acknowledgment This experiment was funded by the Ministry of Research, Technology, and Higher Education through the Program Magister Doktor Sarjana Unggul (PMDSU) in the year of 2016, with contract number: 180/SP2H/LT/DRM/III/2016. References Alba-Hurtado, F., Munoz-Guzman, M.A., 2013. Immune responses associated with resistance to haemonchosis in sheep. Biomed Res. Int. 2013, 1–11. Andriyanto, Amrozi, Rahminiwati, M., Boediono, A., Manalu, W., 2017. Optimum dose and time of pregnant mare serum gonadotropininjections in Kacang goats to increase endogenous secretion ofestrogen and progesterone without superovulation response. Small Rumin. Res. 149, 147–153. Arif, R., Andriyanto, Winarto, A., Boediono, A., Satrija, F., Manalu, W., 2018. Morphometric and Histometric Development of Uterus at the End of Embryonic Stage of Pregnancy in Does Injected With Gonadotropin Prior to Mating. Submitted to Small Ruminant Research. . Behm, C.A., Ovington, K.S., 2000. The role of eosinophils in parasitic helminth infections: insights from genetically modified mice. Parasitol. Today 16, 202–209. Fowden, A.L., Forhead, A.J., 2009a. Endocrine regulation of feto-placental growth. Horm. Res. 72, 257–265. Fowden, A.L., Forhead, A.J., 2009b. Hormones as epigenetic signals in developmental Programming. Exp. Physiol. 94, 607–625. Fowden, A.L., Forhead, A.J., Coan, P.M., Burton, G.J., 2008. The placenta and intrauterine programming. J. Neuroendocrinol. 20, 439–450. Ginting, S.P., Batubara, A., Romjali, E., Rangkuti, M., Subandriyo, 1999. Responses of two genotypes of lambs on the infection of Haemonchus contortus and the level of energy supplements. J. Imu Ternak dan Vet. 4, 20–27. Lacroux, C., Nguyen, T.H.C., Andreoletti, O., Prevot, F., Grisez, C., Bergeaud, J.P., Gruner, L., Brunel, J.C., Francois, D., Dorchies, P., Jacquiet, P., 2006. Haemonchus contortus (Nematoda: trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response. Vet. Res. 37, 607–622. Luffau, G., Khang, J., Bouix, J., Nguyen, T., Cullen, P., Ricordeau, G., Carrat, C., Eychenne, F., 1990. Resistance to experimental infections with Haemonchus contortus in Romanov sheep. Genet. Sel. Evol. 22, 205–229. Manalu, W., Sumaryadi, M.Y., 1998. Maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition in Javanese Thin-Tail ewes with
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