INVESTIGATION OF THE EFFECTS OF LIVE WEIGHT AND GENDER ON

OXIDATIVE STRESS/ANTIOXIDANT PARAMETERS IN HEALTHY GEESE

Abstract

This study was conducted to determine the effects of body weight and gender on
oxidant/antioxidant status in clinically healthy geese. Total antioxidant level (TAS), total
oxidant level (TOS), oxidative stress index (OSI), ferric ion reducing agent in serum samples
taken from a total of 41 geese, 18 males and 23 females, 9 months old, which were bred free-
roaming by the public. antioxidant power (FRAP) and Glutathione (GSH) levels were
measured. It was determined that body weight and gender had an effect on TOS and FRAP
parameters, but did not affect TAS, OSI and GSH values in geese. In the study, a negative
relationship was found between body weight gain and TOS levels. It was detected that FRAP
levels were higher in female geese than in males (p<0.05). As a result; It was concluded that
the relationship between TOS level and body weight in healthy geese should be investigated
in more detail, and that FRAP level could be evaluated as an indicator of antioxidant power in
healthy geese of different genders.

Keywords: Goose, body weight, gender, oxidative stress, antioxidant parameters

Introduction

In our country, goose breeding is commonly carried out in small-scale family

businesses with traditional feeding methods. Gchicks in the conventional feeding method; In
the first week, it is fed with milk, raw eggs mixed with milk, soaked stale, table scraps, mixed
feeds prepared for other poultry. In the following weeks, a very important part of the feeding
is based on pasture (Arslan, C. and Tufan, 2011).

Oxidative stress is defined as a deterioration in the equilibrium state of pro-oxidant

and antioxidant systems in favor of pro-oxidation (Dopsaj et al., 2011). In oxidative stress
situations, the rate of formation of reactive oxygen species (ROS) exceeds the speed of the
antioxidant defense system (Kunsch and Medford, 1999). ROS, which are produced as
natural byproducts of oxygen metabolism, have important roles in cell signaling and
homeostasis under normal conditions. (Devasagayam et al., 2004).

Under normal conditions; cells are protected from ROS by antioxidant defense
mechanisms including enzymes such as superoxide dismutase (SOD), catalase and glutathione
peroxidase (GPx). These antioxidant enzymes are crucial for the normal redox balance in
cells. When this redox balance is disturbed in favor of ROS, oxidative stress and cell damage
occur (Kunsch and Medford, 1999).

Since oxidative stress plays a role in many diseases that occur differently in males and
females, the relationship between gender and oxidative stress is important. It has been
reported that there may be a difference in the expression and/or activities of antioxidant
enzymes between males and females (Kander et al., 2017). It has been reported that oxidative
stress is higher in male rats than in female rats. (Barp et al., 2002). In a different study, in
vivo biomarkers of oxidative stress were found to be higher in young males than in females of
the same age (Ide et al., 2002). These studies show that females are less sensitive to
oxidative stress than males, and there is a significant relationship between gender and
oxidative stress.

Various markers and methods are used to evaluate oxidative stress and antioxidant
status. Since measuring these markers separately is both time-consuming and costly, it has
become more common in recent years to measure total oxidant (TOS) and total antioxidant
status (TAS) and calculate oxidative stress index (OSI) (Irak et al., 2018). The ferric ion
reducing antioxidant potency (FRAP) test, a simple and automated test that measures the
ability to reduce iron, is also offered as a new method for assessing "antioxidant potency"
(Benzie and Strain, 1999). In addition, glutathione (GSH) is an important marker in the
measurement of antioxidant potential (Al-Farnwany et al., 2019).

As far as it is known, there is no study investigating the effects of body weight and
gender on oxidative stress/antioxidant parameters in healthy geese. In this study, it was aimed
to determine the relationship between body weight and gender and oxidant/antioxidant status
by measuring serum TAS, TOS, FRAP, GSH levels and calculating OSI in healthy geese of
the same age, care and feeding conditions.

Material and Methods

The material of this study consisted of a total of 41 geese, 18 male and 23 female, 9
months old and clinically healthy, which were bred free-roaming in the hands of the public.
After a 12 h fasting period of the geese, blood samples were taken from V. subcutanae ulnaris
into tubes without anticoagulant. After separating the serums, the samples were preserved at -
20 O C degrees until analyzed.

Ethical Approval
 This study was approved by Harran University Animal Experiments Local Ethics
Committee (Session and decision number: 2021/009, 01-11).

Measurement of Total Antıoxıdant Status (TAS)

TAS levels were measured using commercially available kits (Relassay, Turkey). The
novel automated method is based on the bleaching of characteristic color of a more stable
ABTS (2,2′-Azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) radical cation by antioxidants.
The assay has excellent precision values, which are lower than 3%. The results were
expressed as mmol Trolox equivalent/L (Erel, 2004).

Measurement of Total Oxidant Status (TOS)

TOS levels were measured using commercially available kits (Relassay, Turkey). In
the new method, oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex
to ferric ion. The oxidation reaction is enhanced by glycerol molecules abundantly present in
the reaction medium. The ferric ion produces a colored complex with xylenol orange in an
acidic medium. The color intensity, measured spectrophotometrically, is then related to the
total amount of oxidant molecules present in the sample. The assay was calibrated with
hydrogen peroxide and the results were expressed in terms of micromolar hydrogen peroxide
equivalent per liter (μol H2O2 equivalent/L) (Erel, 2005).

Determination of Oxidative Stress Index (OSI)

The ratio of TOS to TAS is accepted as the oxidative stress index (OSI). For
calculation, the obtained unit of TAS was converted to μol/L, and the OSI value was
calculated according to the following Formula: OSI (arbitrary unit) = TOS (μol H2O2
equivalent/L) / TAC (μol Trolox equivalent/L) (Harma and Erel, 2003).

Measurement of the FRAP

The determination of serum FRAP levels were performed using a microplate reader
according to the method of Benzie and Strain (1999). The method measures the ability of
antioxidants contained in a sample to reduce ferric (Fe3+) ions to ferrous (Fe2+) ions. This
reduction at a low pH causes a colored ferrous-tripyridyltriazine complex that absorbs light at
λ = 593 nm. The results were expressed as μmol/L.

Glutathion (GSH) assay

 Glutathion (GSH) level was assessed through reaction with OPA (1mg/ml o-
phthaldialdehyde in methanol) following the modified technique of Koyuncu et al. (2017).
GSH was used as a standard. GSH samples were assessed via microplate reader, with
excitation at 345 nm and emission at 425 nm. Results were expressed as nmol/ml in serum.

Statistical Analysis

The data were analyzed with a factorial model of the general linear model procedure
using SPSS software (SPSS version 23.0; IBM Corp., Armonk, NY, USA). The interaction of
live body weight (LBW) and gender on TAS, TOS, OSI, FRAP and GSH parameters were
determined using the PROC GLM procedure.
Yijk = µ + LBWi + Gj + (LBW × G)ij + eijk
Where: Yijk is the response variable (TAS, TOS, OSI, FRAP and GSH); µ is the overall mean
common to all observation; LBWi is the fixed effect of live weight (i = 4); Gj is the fixed
effect of gender (j = 2), (LBW × G)ij is the first order interaction and eijk is the random residual
error. Statistical significance was set at p ≤0.05. Post hoc tests were performed, using the
Duncan's Multiple Range Test.

Result
The serum TAS, TOS, OSI, FRAP and GSH levels of male and female geese with
different body weights are presented in Table 1. It was detected that the live weights of the
geese only affected the TOS value (p<0.05). The highest TOS value (16,383 μmol H2O2
equiv./L) in the study was in geese with the lowest body weight (2.60-2.89 kg), the lowest
TOS level (11.575 μmol H2O2 equiv./L) was in geese with the highest body weight (3.50 kg
and up) were measured (p<0.05). It was seen that the interaction of Gender and Gender x
Body Weight did not affect TOS values (P>0.05). Gender was found to affect only FRAP
level in geese. FRAP levels measured in female geese were found to be statistically higher
than males (p<0.05). It was identified that body weight and Gender x Body weight interaction
had no effect on FRAP levels (P>0.05).
It was detected that body weight, gender and Gender x Body Weight interaction did
not affect TAS, OSI and GSH values (P>0.05).
Table 1. Serum TAS, TOS, OSI, FRAP and GSH levels of male and female geese with
different live body weights.
TAS TOS OSI FRAP GSH
(mmol (μmol (%) (μmol/ (nmol/
 Live body trolox H2O2 L) ml)
 Group weights Equiv./L) Equiv./L)
Female 2.60-2.89 kg 1.367 18.267 1.353 8.887 9.536
  2.90-3.19 kg 1.100 14.390 1.429 7.665 10.295
  3.20-3.49 kg 1.383 15.700 1.137 8.760 10.069
  3.50 and up 1.114 11.750 1.094 11.475 13.952
Male 2.60-2.89 kg 1.424 14.500 1.071 6.627 9.086
  2.90-3.19 kg 1.067 14.213 1.421 7.127 11.523
  3.20-3.49 kg 0.974 13.700 1.680 8.632 10.188
  3.50 and up 1.348 11.400 0.873 7.895 9.488
SEM   0.171 1.323 0.270 1.046 1.103
Live Body Weight
2.60-2.89 kg   1.395 16.383a 1.212 7.757 9.311
2.90-3.19 kg   1.083 14.302a 1.425 7.396 10.909
3.20-3.49 kg   1.178 14.700ab 1.409 8.696 10.129
3.50 ve yukarı   1.231 11.575b 0.983 9.685 11.720
SEM   0.121 0.936 0.191 0.740 0.780
Gender
Female   1.241 15.027 1.253 9.197 10.963
Male   1.203 13.453 1.261 7.570 10.071
SEM   0.088 0.677 0.138 0.535 0.565
Source of Variation        
Gender 0.764 0.113 0.968 0.042 0.275
Live Body Weight 0.298 0.047 0.428 0.182 0.269
Gender * Live Body Weight 0.296 0.500 0.365 0.410 0.152

Discussion

Disruption of the balance between the antioxidant defense system and ROS production
is defined as oxidative stress. Oxidative damage can lead to cellular dysfunction and
contribute to the development of a wide variety of conditions in poultry such as inflammation,
ischemia-reperfusion injury, aging process (Halliwell and Gutteridge, 1983; Ahmed, 2006;
Aydilek et al., 2012).

It is shown that excessive free radical production, weakening of the antioxidant

defense system and oxidative stress are among the main causes of the harmful consequences
of stress in poultry (Surai et al., 2019). The practice of feed restriction in chickens leads to
chronic hunger and stress. It has been reported that some physiological differences (plasma
corticosterone levels, body temperature and heart rate) between ad-libitum and restricted-fed
chickens may be due to differences in metabolic rate as well as differences in stress level (De
Jong et al., 2002). In addition, neuroendocrine peptides and cytokines released mostly from
adipose tissue have been found to play a role in both short-term and long-term energy
balance, metabolism and inflammatory response. (Arslan, N. et al., 2010).

In general, there are technological, environmental, nutritional, biological/internal

stress factors in the poultry industry that cause detrimental changes at molecular/cellular and
physiological levels and ultimately reduce the productivity and reproductive performance of
commercial birds (Surai et al., 2019). Domestication and genetic selection for rapid growth,
improved feed conversion and higher egg production rates have sensitized poultry, including
broilers, layers and turkeys, to oxidative stress (Soleimani et al., 2011). In poultry, the stress
response may occur in different time periods depending on the conditions. It is recommended
that a wide variety of protective systems against oxidative stress be tightly regulated
(Pomatto and Davies, 2018).

While feed restriction can be a source of stress for birds, feeding ad libitum can have
negative consequences for health and therefore animal welfare. It has been shown that there
are differences in physiological stress parameters between the restricted-fed broilers and the
adjubitum-fed control group (De Jong et al., 2002). Metabolic process changes are observed
in nutrient restrictions. Especially the defect caused by hunger in energy metabolism can
cause oxidative stress (Ekizoğlu, 2008).

Within a given species, individuals of the same gender and age often differ from each
other in their behavior and physiology, even under standard conditions. In the study, in which
physiological patterns as well as behaviors were determined in a herd of free-roaming gray
geese living in semi-natural conditions, it was concluded that the levels of corticosterone and
testosterone metabolites excreted in feces may be very important factors of personality in
geese (Kralj-Fišer et al., 2007). In the same study, it was hypothesized that a goose fed in the
middle of the herd under high-density feeding conditions is restricted in free movement and
may be more stressed than a goose fed quickly at the edge of the herd.

In this study, it was determined that the TOS levels of geese with a body weight of
2.60-2.89 kg were higher than that of geese with a body weight of 3.50 kg and above
(p<0.05). It is thought that this situation may be caused by the individual behaviors,
physiology and metabolic differences of the geese.

Differences in susceptibility to oxidative stress have been reported between males and
females Proteggente et al. (2002) They found that the level of oxidative damage to DNA was
higher in male than in female. Some in vitro studies have shown that estrogens have important
antioxidant properties (Subbiah et al., 1993; Ruiz-Larrea et al., 1997). Kander et al.
(2017) It is reported that the oxidative stress differences between males and females may not
be due to estrogen alone, but more than one mechanism may be responsible for the formation
of oxidative stress. TAS, FRAP and GSH are parameters that show antioxidant levels in
serum (Al-Farnwany et al., 2019; Çelik et al., 2019). FRAP analysis demonstrates the ability
of the antioxidant in the sample to inhibit the oxidative effects of the reactive species. Unlike
other tests that measure total antioxidant power, the FRAP test is fast, simple, effective and
economical (Benzie and Strain, 1999).

In a study conducted to compare the antioxidant capacity of heart, kidney, liver and
brain tissues of male and female rats, it was determined that the FRAP level was higher in
female rats than in male rats (Katalinic et al., 2005). It has been reported that different
antioxidant systems show higher activity in females for liver and heart tissue (Barp et al.,
2002; Bureau et al., 2003). In some studies (Persky et al., 2000; Busserolles et al., 2002;
Bureau et al., 2003) it has been reported that estrogen has a direct protective effect against
oxidative damage in heart and liver tissues. Choy et al. (2000) In the study investigating the
total antioxidant activity in human tears, they found FRAP levels to be 463 ± 191 μM in
females and 376 ± 135 μM in men. In this study, FRAP level was found to be higher in
female geese (9.197 μmol/L) compared to males (7.570 μmol/L), which is consistent with
previous studies.

Conclusion

In this study, geese with high body weight were found to have lower TOS levels
compared to geese with low body weight. It is thought that the mechanisms underlying this
situation should be investigated with more detailed studies. In addition, it was concluded that
gender had an effect on FRAP levels in healthy geese and could be evaluated as an indicator
of gender-related antioxidant power.

Acknowledgment
References