IMPACT OF ASCORBIC ACID AND PROBIOTICS FOR IMPROVEMENT OF CONCEPTION RATE OF EWES DURING SUMMER SEASON

IMPACT OF ASCORBIC ACID AND PROBIOTICS FOR IMPROVEMENT OF CONCEPTION RATE OF EWES DURING SUMMER SEASON

HOWIDA M.A. ABD-EL-RAHMAN^* and MAHA A. IBRAHIM^**

^* Field Investigation Department, Animal Reproduction Research Institute, Giza, Egypt.

^** Biology Reproduction Department, Animal Reproduction Research Institute, Giza, Egypt.

   maha_doctor2013@yahoo.com

INTRODUCTION

Heat stress is a very common in livestock especially in tropical countries and sub-tropical areas. Indigenous sheep tends to breed throughout the year, although the sexual activity is restricted to a certain extent to the summer months (Marai et al., 2004). In such regions, a high ambient temperature is the major constraint on animal productivity (Marai et al., 2000; Shelton, 2000). This effect is aggravated when heat stress is accompanied by high ambient humidity (Abdel-Hafez, 2002). Exposure of sheep to elevated temperatures results in a decrease of body weight, average daily gain (ADG), growth rate and body total solids which is reflected by impaired reproduction (Shelton, 2000 and Abdel-Hafez, 2002). Moreover, stressful conditions. such as exposure to heat .Leads to increased free radicals production (Loven, 1988). The elevation of free radicals affects strongly all body systems including reproductive performances of both male and female. Free radical have been suggested to be partly responsible for the detrimental effect of elevated temperature on cellular membrane integrity, and for compromising the cellular function of steroidogenic tissues and embryos, as these have been found to be sensitive to free radical damage (Hansen 1997; Arechiga et al., 1998). A major strategy to reduce the effect of heat stress on animals is to alter the environment through the use of sheds, fans or evaporative cooling (Bucklin et al., 1991). Such practices are not possible in semi-intensive systems as sheep are grazed in the open during most of the day. This necessitates other strategies to counteract the adverse effects of heat stress such as supplementation of antioxidants. An antioxidants such as Vit C is a free radical scavenger, that protects the body defense system against excessively produced free radicals during heat stress and stabilizes the health status of the animal. Although ruminants can synthesize Vit C (McDowell, 1989), a great reduction in plasma Vit C concentration was reported  in heat- stressed cows (Padilla et al., 2006). Vit C may also be able to regenerate other antioxidants such as Vit E (Bruno et al., 2006). The manipulation of rumen microbial activity including dietary antibiotics and probiotics has been widely studied during the last 20 years. The probiotics (bacterial and yeast cultures) are live microbial feed supplements that have been used as growth promoters to replace the widely used antibiotics and synthetic chemical feed supplements (Sumeghy, 1995 and Strzetelski, 1996). Dawson (1990) reported that, yeast culture increased ruminal cellulose digestion and consequently improved feed efficiency and gain and increased the microbial growth in rumen and enhanced the microbial protein synthesis. Dawson (1994) showed that, yeast or yeast culture are rich source of vitamins, enzymes and other important nutrients and co-factors which make them attractive as digestive enhancers as a basic source of nutrients. El-Shaer (2003) reported that, all nutrients digestibility were increased for sheep. Also, improve nitrogen balance (El-Ashry et al., 2003). Abdel-Latif (2005) found an increase in total VFA due to adding yeast culture in sheep diets. Saccharomyces cerevisiae were reported to balance the energy and the acid-base metabolism in sheep that resulted in a significantly higher milk production and increasing milk yield of ewes which is an important factor for the production of robust lambs at weaning (Helal and Abdel-Rahman, 2010). During heat stress, sheep performance was improved by adding YC, El-Shaer, (2003) detected a decrease in rectal and skin temperatures as well as pulse and respiration rates. Also, an improvement in reproductive performance was also obtained by Abdel-Khalek (2003) in Friesian cows and by Ebrahim (2004) in Egyptian buffaloes due to yeast culture supplementation. 

Aim of the work:

It was conducted to assess whether supplementation of vitamin C(Vit C) or live dried yeast (DY/probiotics) could alleviate heat stress drastic changes in ewes or not.

MATERIALS and METHODS

The present study was carried out at the experimental farm belonging to Animal Reproduction Research Institute (ARRI). Twenty four sexually mature Barki ewes of 2-3 years of age and average weight 30-34 kg confirmed by ultrasound examination(knorton, 6.5MC, sigma 300, France) that they are non pregnant before the start of the trial. Ewes were housed under conditions of natural day light and temperature in semi-shaded well ventilated pens. Ewes were offered basal ration according to management of ARRI and water was offered ad libitum. A second ultrasound scanning for pregnancy detection was performed one month after ram exposure (natural mating). Ewes were randomly divided into 3 groups, each of 8 animals. The control group fed the basal ration without any supplementation [group (1)]. In treatments viamin C (Vit C) was given at 75/mg/kg BW/d (powder supplied by the Egyptian Arabian Co. of feed additives and vitamins) [group (2)]. Live Dried Yeast (DY) was supplemented at 5/ g/head/day (Yea-Sacc 1026, Saccharomyces Cerevisiae) [group (3)]. Vit C and DY were supplemented to the animals orally through the period of the experiment. The treatments were given 2 weeks premating and lasted for 4 weeks postmating during the summer season (June to August) where the highest temperature level recorded in Egypt (figure 1) describes temp /humidity level in this period.

All ewes were weighed before and after the end of the treatment. Respiration rate (RR) and rectal temperature (RT) of the ewes were recorded in the three groups in the period before the treatment and then regularly at 2 weeks intervals till the end of the treatments. Rectal temperature was measured using digital thermometer. After 2 weeks from the beginning of the experiment all ewes were subjected to estrus detection 2-times daily by good vasectomies ram. Conception rate of ewes in the three groups was recorded by ultrasonography at 4 weeks post examination.

Blood samples were collected from all ewes regularly at 2 weeks intervals from the beginning of the trial (on the second, fourth and six week), ten ml of blood was collected from jugular vein before feeding. Blood was collected in two vaccutainer tubes one containing EDTA for plasma and the other plain for serum separation. Serum and plasma were obtained by centrifugation at 3000 rpm/15 min samples aliquoted and frozen at -20 C until analysis. Blood samples were analyzed colorimetrically by commercial kits for: Total antioxidant capacity (TAC) (koracivic et al., 2001), ascorbic acid (Vit C) (Harris and Ray, 1935), Glutathione - S- transferase (GST) (Habig      et al., 1974). Free radicals: Lipid peroxidase [malondialdehyde (MDA)] (Ohkawa et al., 1975) and Nitric oxide (NO) (Rajaraman et al., 1998).

All hormones were performed using ELISA microwells kits (Monobind Inc. Lakeforest, CA 92630.USA): Total triiodothyroxine (T₃): Braverman, (1996), total thyroxin (T₄): Muzzaffari and Gharib (1998), cortisol: Burtis and Asweed (1994).

Statistical analysis:

All data were subjected to statistical analysis according to Snedecor &Cochran (1982) and results were expressed as Means ± standard error (SE). Differences between means in different groups were tested for significance using T test as independent t test for all parameters except for thermo-respiratory activities were tested for significance using paired "t" test.

RESULTS

The overall conception rate of control and treated groups are illustrated in Figure 2, that showed that there is a highly significant (P < 0.01) increase in conception rate (100%) due to Vit C supplementation as compared with that of control one (62.5%). Meanwhile the same figure clearly indicated that conception rate was not affected by DY supplementation (62.5 %) as compared with that of control group.

The results of thermo-respiratory activities are shown in Table (1), revealed that the Vit C and DY treated groups had a significantly (P≤0.01) lower rectal temperature and respiratory rate values as compared with the pre-treatment values. On the other hand, there was a nonsignificant change in rectal temperature and respiratory rates in the control group throughout the trial, also, average rectal temperature and respiratory rates values before treatment were similar in the control and treated groups

The data in Table (2) showed that there is a significant (P <0.05) increase in Vit C and glutathione -S- transferase (GST) levels in treated groups (1&2) than the control one. While, total antioxidant capacity (TAC) serum level showed a significant (P <0.01) increase in both treated groups during the 6^th week only when compared with that of the control one.

The data in Table (3) showed that there is no difference between all experimental groups in nitric oxide (NO) serum level all over the trial; however, there was a significant decrease in malondialdehyde (MDA) level in both supplemented groups after the 6 ^th week of the treatment as compared with that of the control one.

With regard to thyroid hormones and serum cortisol levels, data in Table (4) clearly indicated that the level of serum T₃ in Vit C group was elevated (P<0.05) significantly throughout the 2^nd and the 6^th weeks and was elevated nonsignificantly throughout the 4^th week of the trial compared to the control one. Meanwhile, its level in DY treated group was elevated (P<0.05) significantly throughout the 2^nd week and nonsignificantly throughout the 2^nd and the 6^th weeks of the trial when compared with the control one. In respect to serum T₄ level which was elevated (P<0.01) significantly throughout the 2^nd and the 4^th weeks and was elevated nonsignificantly throughout the 6^th week of the trial in Vit C and DY  treated groups as compared with the control one.

In respect to serum cortisol level which was significantly (p<0.05) decreased in Vit C treated group throughout the trial, meanwhile the decrease in serum cortisol level due to DY supplementation was significantly (P<0.05) only during the 2 ^nd week and nonsignificantly during the 4 ^th and 6^th  weeks of  the treatment as compared with control group. 

The change in body weight due to supplementation and heat stress in the present experiment are presented in Table (5). Data analysis showed that supplementation of Vit C to heat stressed ewes prevent weight loss due to heat stress and significantly (P < 0.05) increased body weight of the treated group throughout the trial as compared with the control one. In the same table the results revealed that DY minimized weight loss due to heat stress and did not improve body weight of the treated ewes as their initial weight was unaltered with DY supplementation, and there was a nonsignificant difference between the DY supplemented group and the control one in respect to body weight throughout the trial.

Figure 1: Cairo,Egypt climate showing high temperature and humidity in summer season.

Figure 2: Conception rate of all ewes after 6 weeks of the trial.

Table 1: Means ± SE of thermo-respiratory activities of the control and treated groups.

 * Significant at P <0.05, ** Significant at P <0.01, *** Significant at P <0.001, ttt: treatment

Table 2: Means ± SE of serum vitamin C (Vit C), total antioxidant Capacity (TAC) and glutathione-s-transferase (GST) concentrations of the control and treated groups.

Means with different superscripts letters in the same row are significantly different at P<0.05 and P<0.01, ttt: treatment, no: nonsignificant

Table 3: Means± SE of serum Nitric oxide (NO), Malondialdehyde (MDA) concentrations of control and treated group.

Means with different superscripts letters in the same row are significantly different at P<0.01. 

Table 4: Means ± SE of serum triiodothironine (T₃), thyroxin (T₄) and cortisol levels of the control and treated groups.

Means with different superscripts letters in the same row are significantly different at P<0.05 and P<0.01, ttt: treatment, ns: nonsignificant. 

 Table 5: Means ± SE of body weight (kg) of the control and treated groups.

Means with different superscripts letters in the same row are significantly different at P<0.05, ttt: treatment, M: month.

DISCUSSION

Our results was clearly indicated that premating dietary Vit C supplementation to ewes during summer season (heat-stressed) significantly improved conception rate as compared with control ewes. This results were in agreement with those obtained by (Haliloglu and Serpek, 2000) who reported that Vit C supplementation to the sheep during the breeding season improved their fertility, that also were in  constant with Marai et al. (2004) who found a  negative relationship between conception rate and ambient temperature in sheep. Such improvement of conception rate in Vit C supplementation group may be attributed to the increment of progesterone secretion and/or protecting the embryos from the harmful effect of free radical produced with heat stress and allowing them to bypass the period when they are most sensitive to elevated temperature (i.e., in 2 ^nd days after breeding), low progesterone level associated with enhanced secretion of PGF2α which may induce luteolysis of the corpus luteum and termination of pregnancy (Santos et al., 2004), Vit C content in the CL is at its maximum when the CL is fully mature, remains high during pregnancy, and decreases as the CL regresses (Petroff et al., 1997). One of the proposed mechanisms to explain the luteolytic effect of PGF2α is the generation of the oxidative radicals, and the cytotoxic effect (Niswender et al., 2000). And it was suggested that the oxidative radicals may be fundamental in the initiation of luteolysis and the consequent inhibition of the progesterone synthesis (Tanaka et al., 2000 and Pepperell et al., 2003). Moreover, it was found that Vit C supplementation to embryo culture medium (IVF) followed by incubation at 20% oxygen level resulted in significantly higher rates of cleavage, morula and blastocyst formation and blastocyst total cell count in sheep, which reflect the protective effects of Vit C against the oxidative damage to DNA (Natarajan et al., 2010). Meanwhile, there is no significantly difference in conception rate between DY supplemented group and the control one. These results were agreed with those obtained by Abd Rahman et al. (2012) and Mousa et al. (2012) who reported that supplementation of yeast had no significant effect on conception rate.

The mean of ambient temperature and relative humidity during the experimental period were illustrated in (figure 1) which was considered as heat stress condition. The exposure to high ambient temperatures, is a major constraint on animal productivity as it evokes a series of drastic changes in the animals’ biological functions, Such changes resulted in the impairment of productive and reproductive performance. The effect of heat stress is aggravated when it is accompanied by high humidity (Marai et al., 2007). Poorly sweating animals like sheep (due to very thick wool fleece) attempt to maintain the heat balance by increasing their respiratory activity, losing more heat through evaporation from the respiratory tract. Our results, revealed that Vit C supplementation minimized the elevation of rectal temperature due to heat stress and significantly decrease rectal temperature of treated ewes than before treatment. These results were in agreement withSivakumar et al. (2010)who found that Vit C supplementation to heat stressed goats significantly decreased the rectal temperature and respiratory rates. Vit C directly alters the thermal set point by decreasing the prostaglandin output, especially of PGE series, whose turnover increases during stress and has a direct effect on the hypothalamic thermoregulatory zone (Ganong, 2001). Similarly, our results indicated that there is a significant decrease in RT, RR values for the DY supplemented group as compared to the control one. These findings are in accordance with those obtained by Abdel Rahman et al. (2012) on sheep who indicated that DY supplementation significantly decreased RT and RR. That may due to minerals content of DY, especially selenium which has a favorable effect on body thermoregulation.

Heat stress stimulates excessive production of free radicals (superoxide anion, hydroxyl, hydrogen peroxide and singlet oxygen) (Bernabucchi et al., 2002) that can damage healthy cells if they are not eliminated. Heat production is directly controlled by the nervous and endocrine systems through the modifications of appetite, digestive process and, indirectly, by alterations of the activity of respiratory enzymes and protein synthesis (Yousef, 1985). As shown in Table (2), dietary supplementation of DY and Vit C during heat stress caused a higher level of total antioxidant that only were significant statistically in Vit C group (p < 0.05). While, glutathione-S-transferase was significantly higher in both treatments. The results of the present study suggested that Vit C supplementation may be beneficial to ewes under heat stress which may alleviate its adverse effects, that agreed with Matsui, (2012); Abdul Kareem (2011) and Padilla et al. (2006) who explained the positive role of Vit C in the oxidative stress as antioxidant by protecting the Red Blood Corpuscles (RBC) membrane (consist of phospholipids) against the harmful oxidation processes and thus reduce the  RBC haemolysis via increasing the flexibility of the cell membranes (so, it maintain the oxygen level and so, the respiratory rate). Dietary supplementation of ewes with DY during heat stress condition can improve oxidant / antioxidant balance under hot conditions that agreed with Aït-Belgnaoui (2009) and Messaoudi et al. (2011) who reported that the oral intake of certain live dry yeast may act in a direct way on the animal well-being by reducing the physiologic response related to stress. They added that there is a two-fold link between probiotics and animal well-being: not only probiotics allow to treat stress consequences (flora balance, transit regulation, strengthening of the ruminal ecosystem, etc.) but they also they  enable to reduce directly the animal stress perception and improving the well-being feeling of the animal. This is all more important as animal well-being is not only a moral and social necessity but it’s an essential condition to have optimal zoo technical performances. Moreover,Maziar et al., 2007 found that the addition of probiotics to diets benefit the host animal by stimulating appetite, improve intestinal microbial balance, stimulate the immune system, decreased pH and release the bacteriocins. In the last few years, the detection of free radicals damage and the protection against it has become very important in the studies related to ruminant production/reproduction as the level of lipid peroxidation (malondialdehyde, MDA) and antioxidant status give complementary information about the metabolic status of the animal rather than metabolic parameters alone (Castillo et al., 2003).

In the present investigation, it was found that probiotics, and Vit C addition decreased lipid peroxidation biomarker (MDA) significantly in both supplemented groups. This is may be referred to vitamins dual effects: (a) control of metabolic pathways or gene expression and (b) Reactive Oxygen Species (ROS) trapping activity (Aurousseau et al., 2006). In addition, Vit C helps to scavenge the oxygen radicals throughout the female reproductive tract (Goud et al., 2008). Although cattle can synthesize Vit C in the liver, a large reduction in plasma Vit C concentration was reported in heat stressed cows (Padilla et al., 2006). The supplementation of ascorbic acid has been shown to enhance the plasma ascorbic acid concentration and immune response of the cattle subjected to environmental stressors (Padilla et al., 2007).

Concerning serum T₃ and T₄ levels in Vit C supplemented ewes there was a significant increase in serum T₃ and T₄   at all weeks of the trial except at the 4^th week which showed a non-significant increase in serum T₃ level and at the 6^th week which showed a non-significant increase in serum T₄ as compared with the control ewes. Marai et al. (2000) confirmed that T₃ concentration in blood plasma was significantly lower in summer season than in winter season in sheep. Decreased thyroid hormone levels during heat stress are an adaptive response and also might be an attempt to reduce metabolic rate and heat production, as thyroid and glucocorticoid hormones assumes a fundamental role in endogenous heat production and regulation(Abeni et al., 2007). This modification in thyroid activity due to heat stress is consistent with decrease in food intake and metabolic heat production (Alameen and Abdelatif., 2012).These results were consistent with those of Sivakumar et al. (2010) who noticed that Vit Csupplementation significantly increased serum T₃ and T₄levels in heat stressed goat and Al- Katib (2001) who found that Vit C cause  an increase in thyroid gland weight and activity. On the other handUsha et al. (2002)related the findings to the free radical H₂O₂ (hydrogen peroxide) that served as a substrate for the thyroperoxidase enzyme which catalyzed the synthesis of thyroid hormones. Production of more H₂O₂ under stress condition might have reduced the levels of thyroid hormones. Moreover, 5' mono deiodinase, an enzyme which converts T₄ to T₃, is affected by free radicals under heat stress (Brzezinska et al., 2001). Increased levels of thyroid hormones in Vit C supplemented group in the present study might be due to protection of the above mentioned enzymes from free radicals. Thus, this increment in serum T₃ and T₄ levels due to dietary Vit C supplementation reflect the alleviation in heat-stress in the treated ewe. However, similar findings were observed and supported by Ghanem, (2005) who stated that Vit C supplementation may alleviate stress in Awassi sheep.

At the same time, the DY group showed an increase in serum T₃ level was significantly at the 4^th week and non-significantly at the 2^nd and the 6^th week, meanwhile due to dietary DY supplementation a significant increase in serum T₄ at all weeks except at the 6^th week which showed only a non-significant increase in serum T₄ level,. These results were agreed with Sarwar et al. (2010) who found that Lambs fed diets with probiotic had higher T₄ level than those fed diets without additives, and a higher T₃ level was also noticed in lambs fed diets containing probiotics than those fed ionophore.

Concerning serum cortisol level, the obtained data indicated there is a significant reduction in serum cortisol level in premating dietary Vit C supplementation group all over the trial, that may be due to the impact of Vit C to reduce the magnitude of hyperthermia caused by heat stress. This finding was in accordance with Sivakumar et al. (2010) who ststed that the  supplementation of Vit C had a negative effect on cortisol levels during heat stress, which relived the severity of heat stress in goats. The blood cortisol level is generally considered as a reliable physiological index for determining the animal response to stress, as a consequence of the activation of the hypothalamic-pituitary-adrenal axis, heat stress causes an increase in cortisol production (Louise, 2003 and Silanikove, 2000). Bouraoui et al. (2002)found a positive correlation between temperature-humidity index (THI) and cortisol concentration that support the rise in cortisol level in the control group in the present study. On the same hand, DY supplementation decreased significantly the serum cortisol level during the 2 ^nd week and non- significantly at the 4^th and the 6^th weeks of the treatment. These results were in accordance with those reported by Messaoudi et al. (2011)who noticed that probiotics directly decreased the cortisol levels by improving the gut flora that affects the brain neurotransmitters that produced in the gut. There is a well established link between stress, central nervous system and gastrointestinal disease. Additionally, probiotis decreases the unhealthy pathogens in the gut helping to get rid of inflammation symptoms and inflammation biomarkers (C- reactive proteins) which lead to less depression (Messaoudi et al., 2011). 

In the present study dietary Vit C supplementation to heat stressed ewes prevent weight loss as compared with the control. This result was supported byHaliloglu and Serpek (2000) who found that Vit C supplementation increased the body weight of pregnant ewes and newly born lambs. A possible explanations for such improvement in body weight, is the elevation of glucose and protein levels Abdul Kareem (2011),thatmay be due to reduction of oxidative stress produced by heat stress (Bruno et al., 2006). Also, Vit C increases food consumption and food conversion and causes an increase in thyroid gland secretion resulted in an increase metabolic rate and thus improves body weight (Al- Katib 2001). In contrast DY supplementation did not improve body weight of the treated ewes as their initial weight. These results are in accordance with those reported by Mukhtar et al. (2010) who reported that the initial, final and daily weight gain, were non significant due to yeast culture supplementation for growing lambs. An opposite trend was reported by komonna (2007) Helal and Abdel-Rahman (2010) who found that addition of yeast culture of diets of ewes during nursing period resulted in improving its feed utilization and resulted in satisfactory ewe live weight and lamb growth rate. The confliction in the results concerning the effect of DY on body weight and weight gain may be attributed to facts indicating that efficiency of probiotics differ depending upon the probiotic dose rate, species and  age of animals, viable yeast cell number, diets composition, strains of yeast and the season of the treatment.

CONCLUSION

The results of the present study revealed that Vit C offers a thermo- protective role that lessen the destructive effects of heat stress in breeding ewes, and improved the conception rate, physiological parameters, antioxidant biomarkers and endocrine responses. So, Vit C can be used as anti-heat stressors to breeding ewes in the premating period as it more economically and favorable than live dried yeast under these conditions.

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