LIPID PEROXIDATION IN FRESH AND STORED SEMEN OF GREEN-LEGGED PARTRIDGE

Agnieszka Partyka¹, Anna Jerysz¹, Przemysław Pokorny^2
1 Institute of Animal Breeding, Department of Poultry Breeding, Wrocław University of Environmental and Life Sciences, Poland
² Department of Limnology and Fishery, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

Study on semen of native Polish breed Green-legged Partridge was carried out. Morphology of spermatozoa and production of malondialdehyde (MDA) in fresh net semen and semen diluted 1:1 with EK diluent and stored for 24 hours at 4°C were examined. Significant differences (P≤0.01) between fresh semen and stored with regard to pH (7.15 vs. 7.40), osmotic pressure (298 vs. 345 mOsmol/kg) and number of total live spermatozoa (97.3% vs. 87.6%), morphologically normal (84.8% vs. 65.4%) and with bent neck spermatozoa (1.6% vs. 12.0%) were observed. Lipid peroxidation expressed as concentration of MDA in fresh semen (0h) and after 24 h of storage did not differ statistically and averaged 0.28 and 0.33 nmol/50 x 10⁶ spermatozoa, respectively.

Key words: Green-legged Partridge, fowl semen, spermatozoa morphology, lipid peroxidation.

INTRODUCTION

The possibility of increasing the efficacy of reproduction in different poultry species and breeds had been investigated and discussed for several years continuously [5, 14, 21]. More and more attention is dedicated to application the artificial insemination procedures in programs of protection wild or endangered birds [4, 10, 19]. Polish fowl Green-legged Partridge as endangered breed demonstrated by FAO was included in “Red Book of Animals”. This breed was distinctioned at the end of XIX century. Nowadays it is kept in conservative breeding flocks, which basic aim is to preserv the genetic resource. One of the great advantages of Green-legged Partridge is an easy adaptation to extensive environmental conditions. Also, utilisation of natural nutrients from green forages contributes to raising taste quality of meat and eggs with lower cholesterol content. These attributes and lower financial expenditures for maintenance Green-legged Partridge in contrary to other poultry species predispose it for keeping in small household or agroturistical farms [20].

Success in artificial insemination depends on many factors, for example: chemical composition of diluent and method of semen storage [25]. Studies on composition of diluents for poultry semen enabling its storage for several hours at low but plus temperatures are performed from a long time [14]. Bird’s spermatozoa show physiological differences and different metabolic requirements. Diluent with suitable chemical composition, pH value and osmotic pressure can provide them conditions maintaining a high reproductive capability during in vitro storage [22]. The ability to obtain fertility result similar to natural mating is caused by the use of diluted semen in artificial insemination [14]. One of the reasons of diverse level of fertilization can be changes occurring in metabolism of spermatozoa. During semen storage, the spermatozoa are exposed to atmospheric oxygen, which can affect peroxidation of lipids contained in their plasma membranes and production of free radical [15, 16, 18, 32].

Lipids are one of the main components of sperm membrane providing them energy and participate in numerous biochemical processes. The lipid and fatty acid composition (saturated : unsaturated) play an important role in sperm function. The high concentration of polyunsaturated fatty acids (PUFAs) within sperm membrane determines peroxidative susceptibility of spermatozoa [15, 30]. Early studies suggested that peroxidation process is started during in vitro storage of semen [9, 32]. Moreover, recently studies indicate that lipid peroxidation occurs during ejaculation [5, 6] and even considerably earlier in the male reproductive tract [24].

An intensity of lipid peroxidation depends on conditions to which spermatozoa are subjected. Previous studies demonstrated different degree of lipid peroxidation in sperm depending on semen diluents [16, 18] and storage temperatures [17, 24]. Malondialdehyde (MDA) production is a result of lipid peroxidation. Its level determines the degree of sperm membranes integrity and their fertilizing ability [1, 24]. Significant correlation between increase in MDA level and decrease in fertility has been shown by Douard et al. [15] and Wishart [32].

Localization of changes and spermatozoa damages occurring during semen in vitro storage is determined by modern methods including evaluation not only external structure but also the ultrastructure of spermatozoa. Except of commonly used eosin-nigrosin staining technique, which allows analysis of sperm morphology with live-dead classification, there are also used methods determining degree of plasma membrane integrity and microstructure of spermatozoa [29]. One of the techniques allowing estimation of sperm membrane damages is reaction of thiobarbituric acid (TBA). Its measure the concentration of lipid peroxidation products reacting with TBA (TBARSs-thiobarbituric reactive substances), including malondialdehyde (MDA). For enhancement the rate of this reaction ferrous ion and ascorbate are used as suitable promoter system [1].

The aim of this study was to determine the malondialdehyde production and changes in spermatozoa morphology in fresh and stored 24 h at 4°C Green-legged Partridge chicken semen.

MATERIALS AND METHODS

Animals
The experiment was carried out with 10 roosters of Green-leged Partridge. Males were kept individually in cages (60x50x75cm) at 18-20°C, under 14L: 10D photoperiod. Birds were fed with a dose of 120-130g/day of commercial feed for breeding flocks, containing 11.7 MJ metabolise energy and 17 g crude protein per kg. Water was provided ad libitum.

Semen collection and treatment
Semen was collected by abdominal massage according to Burrows and Quinn [7] and then polled. Lipid peroxidation, pH value (pH-meter P 731), osmotic pressure (mOsmol/kg, Semi-Micro Osmometr-Knauer), spermatozoa concentration (by haemocytometer method) and morphology (under light microscope Jenaval Carl Zeiss Jena, Germany) were examined in fresh (0h) and semen diluted 1:1 with EK extender [26] after 24 h storage at 4°C. Morphology of the spermatozoa was evaluated in smears stained by eosin-nigrosin. In each smears 300 spermatozoa were observed. Histological smears were prepared on warm plate at 37.5°C (Bio-Optica Milano SpA). Twenty semen collections and analysis were made.

Determination of MDA concentration
Lipid peroxidation level was examined by measuring the production of malondialdehyde (MDA) according to compilation of Bartosz [3], Castellini et al. [8] and Aitken et al. [1] methods.

Two volumes containing 50 x 10⁹ spermatozoa each (blank trial and sample) was taken and then completed to 1 ml with EK diluent. Samples were incubated in the presence of 0.25 ml ferrous sulphate (0.2mM) and 0.25 ml sodium ascorbate (1mM) for 1hours at 37°C to enhance lipid peroxidation signal. After incubation 1 ml of 15% trichloroacetic acid (TCA) to blank trial and sample, 1 ml of 0.375% thiobarbituric acid (TBA) only to sample and distilled water to control sample was added. Zero sample was created with 1 ml distilled water, 1 ml of TCA and 1ml of TBA. After that, all specimens were boiled for 10 min at 100°C in water bath (MLL 547 AJL Electronic). The precipitate was removed by centrifugation at 5000 x g (centrifuge MPW, 337) for 10 min at 4°C and the absorbance was determined at 532 nm (Spectrophotometer Carry Conc UV-VIS). MDA concentration was defined according to formula:

C = A_sample – A_s.control / ε × 1cm
ε = 156 mmol^-1 x 1 x cm^-1

Chemicals
Nigrosin water soluble (N4754), eosin Y (119830), sodium citrate (S1804), 2-thiobarbituric acid (T5500), trichloroacetic acid (T9159), were purchased from Sigma Chemical Company. Hydrochloric acid (0.1N HCl), sodium ascorbate and iron (II) sulphate were obtained from POCH.

Statistical analysis
The data obtained were presented as means ± SD and analysed by ANOVA and Duncan’s multiple range test. Percentages were transformed by arcsine.

RESULT

Sperm traits
The most of semen traits analysed in fresh (0h) and stored semen at 4°C (24h) differed significantly (P≤0.01).

In freshly collected semen concentration of spermatozoa varied from 2.4 to 4.8 x 10⁹ ml^-1, pH between 6.97 and 7.44 and osmolarity amounted from 295 to 300mOsmol/kg. In diluted and stored semen both, pH value and osmotic pressure increased (from 7.24 to 7.54) (from 300 to 390mOsmol/kg), respectively (Tab. 1).

Morphological assessment of sperm in fresh semen showed average 97.3% of total live spermatozoa, 84.8% of live morphologically normal cells and about 12% deformed spermatozoa. Among live deformed cells, spermatozoa with bulb head and bent neck were the most frequently observed (Tab. 1, Fig. 1).

Semen storage decreased the percentage of total live spermatozoa (88% on average) and morphologically normal sperm (65%), while the amount of spermatozoa with bent neck increased by 10 percent points and dead cells by 10 percent points comparing to freshly collected semen. Percentage of bulb head sperm remained on the constant level (Tab. 1, Fig. 1).

Table 1. Table 1. Characteristics of fresh (0h) and stored 24h at 4ºC semen of Green-legged Partridge, (n=20; ± SD)

Lipid peroxidation
Lipid peroxidation expressed as concentration of malondialdehyde, varied from 0.097 to 0.482nmol/50 x 10⁶ spermatozoa in fresh semen (0h) and from 0.137 to 0.575nmol/50 x 10⁶ spermatozoa in semen diluted and stored for 24 h (Tab. 1).

DISCUSSION

In vitrostorage of poultry semen affects spermatozoa motility, viability and morphology [18, 25, 5, 6, 15, 16] Therefore, semen evaluation after its storage is an important factor for artificial insemiantion practice.

The values of fresh semen obtained in the present study, such as spermatozoa concentration, pH and osmotic pressure were on the physiological level. Similar values of sperm this fowl breed were observed by Niewierska [28]. In the diluted and stored semen, these characteristics increased significantly (P≤0.01). Lake and Ravie [23] described the variation in pH value during storage of diluted fowl semen.

Eosin-nigrosin staining method allows distinguishing morphologically normal spermatozoa from abnormal or dead sperm. In microscopic image of fresh semen (0h) average values of total live sperm (97%) and morphologically normal sperm (85%) were similar – 96% and 80%, respectively- to our earlier result (data not published). Definitely, lower number (68%) of normal spermatozoa in semen of the same breed was observed by Niewierska [28]. However, comparable content of this morphological form in chicken semen was reported by Blesbois et al. [6]. The average percent of deformed spermatozoa in Green-legged Partridge semen amounted 12% and was almost two times lower than in experiment of Niewierska [28], whereas Danikowski et al. [13] reported slightly lower content of this sperm form (10%). Among all deformed cells in the fresh semen, bulb head and bent neck (64% and 13%, respectively) were the most frequently observed. Similar phenomenon was showed by Niewierska [28] for Green-legged Partridge, Chełmońska et al. [11, 12] in quail semen, Maeda et al. [27] in duck semen and also by Łukaszewicz and Kruszyński [26] in gander semen. Thus, it can be suppose that these two morphological forms belong to main deformities of bird spermatozoa.

Analysis of stored semen showed significantly (P≤0.01) reduction in amount of total live (by 10 percent points) and morphologically normal spermatozoa (by 20 percent points) in relation to the fresh semen. Simultaneously, the amount of deformed cells increased by 10 percent points and in it, sevenfold increase of bent neck spermatozoa was observed. Similar results were obtained Łukaszewicz [25] for laying-type chicken.

Malondialdehyde (MDA) is a metabolic breakdown (by-product) product of polyunsaturated fatty acid (PUFA) oxidation. Bird spermatozoa characterise high content of phospholipids, containing polyunsaturated fatty acid residues [30, 31]. They are particularly susceptible to lipid peroxidation, affecting infertility of males. From physiological point of view, a high proportion of PUFA in poultry spermatozoa is important for maintenance membrane fluidity and flexibility, being crucial propriety during fertilisation process [19]. Fujihara and Howarth [17] confirmed opinion that sperm exposed to oxygen during storage, undergo peroxidation. Long and Kramer [24] stated as the paradox that the same oxygen, which is indispensable for sperm metabolism, is then corporate in lipid peroxidation and production free radicals during storage of bird semen. The knowledge of malondialdehyde production during sequential steps of semen conservation process is useful, because can help in spermatozoa prevention against damage during this process and maintenance their fertilizing ability.

In this study both, in freshly collected semen and in semen diluted and storaged 24 h at 4°C, lipid peroxidation expressed by production of MDA was detected. In fresh semen (0h) MDA value equalled 0.28 nmol/50 x 10⁶ spermatozoa (5.6 nmol/10⁹ spz). Considerably lower level of peroxidation in rooster sperm (0.02 – 0.04 nmol/10⁹ spermatozoa) was noted by Blesbois et al. [5]. However described changes were observed in freshly ejaculated semen, thus in the only first step of semen preparation for storage, what from physiological point of view seems to be normal phenomenon. Danikowski et al. [13] reported MDA production in pooled rooster semen amounting 3.37 nmol/ml, but authors did not inform how many spermatozoa were in 1 ml of semen.

The level of MDA production depends on diluent applied, time and temperature of storage [16, 17, 18, 24] In the present study in diluted and 24h stored semen average value of MDA was 0.33 nmol/50 x 10⁶ spermatozoa (6.70 nmol/10⁹ spz). Although malondialdehyde concentration in storaged semen was 1.18 times higher than in freshly collected, obtained differences were not confirmed statistically. It seems that this advantageous result can be affected both, by the choice of adequate diluent and its rapid protective action for the spermatozoa. According to Douard et al. [16], already the first hour of storage is important with regard to lipid peroxidation because in this time authors observed MDA production remaining on stable level for next 48 hours of storage. Our suppositions are partly confirmed by results of Fujihara and Koga [18] and Suray et al. [30]. They found that some volumes of sperm plasma, diluents and also chemical reagents, like calcium or citrate have significant properties to inhibit lipid peroxidation in sperm. Absolutely different results were obtained by Long and Kramer [24], which reported threefold increase of MDA production in turkey semen storaged in analogous conditions in comparison to fresh semen. It has to be pointed that information of Fujihara and Howarth [17], who obtained fourfold lower level of sperm peroxidation comparing to our results, but at temperature reduced to 0°C.

Production of malondialdehyde depends not only on above-mentioned factors. For its concentration in the semen, have also influence males, which produced higher volumes ejaculates and also with high value of MDA. Wishart [32] informed about individual rooster differences where MDA concentration oscillated between 0 to 8 nmol/10⁹ spermatozoa. Also Long and Kramer [24] noted impact of individual turkey on lipid peroxidation (from 0.7 to 1.2 ľmol/50 x 10⁶ spermatozoa). While Douard et al. [15] reported that lipid peroxidation in sperm during in vitro storage is correlated positively with age of males.

CONCLUSIONS

Analysing presented results, it is necessary to deliberate that components of diluent used might be add to antioxidative system protecting spermatozoa against damages during in vitro storage and contribute to inhibition endogenous lipid peroxidation. Because as it was showed by our results elongation of in vitro storage time after dilution with EK in lower temperature 4°C did not affect on significant increase of MDA concentration. Probably, there appeared beneficial interaction between semen, diluent and temperature. It contributed to make advantageous conditions for the sperm. Our presumptions are partially documented by Fujihara and Koga [18] and Surai et al. [30].

In refer to sperm structure, against discussed literature as well as our studies it seems to that cause of significant (P≤0.01) differences in sperm morphology between fresh collected semen and stored semen, might be increase of osmotic pressure and pH. Dependence between pH and deformed and dead spermatozoa is reported by Danikowski et al. [13]. However, it is necessary to think, why in the semen stored 24 hours significant (P≤0.01) increase of dead spermatozoa did not cause significant increase of MDA production comparing to fresh semen, and mutual correlation. Perhaps the answer to this question we could obtain after study of sperm morphology and level of lipid peroxidation after the first hour of storage. How previously mentioned, this time is important from point of view of MDA production [16]. It is supposed to that number of deformed sperm especially dead was not so large to significantly increased level of MDA. Moreover, is suggested that it is necessary used other technique or examined MDA in only spermatozoa. Many questions remained for which we can answer after carry out next studies on this breed of roosters.

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Accepted for print: 29.03.2007

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