Different estrous synchronization systems exist for ewes, which is used to increase the productiveness percentages. De K et al. (2015) conducted a study to assess the success of estrous synchronization in sheep under field conditions of a semi-arid tropical region. A total of 471 ewes were synchronized for estrous during the years 2011 and 2012, the conclusion encourage the use of estrous synchronization for genetic improvement and economic benefit that can be realized by its use. It is very common today to use synthetic progestogens, such as medroxy progesterone acetate (MPA) for a period of 8 to 12 days, which have proven effective for synchronizing estrus in ewes Martínez-Priego and Torres (2007); Dias et al. (2015) and Manes et al. (2015). In addition, application of gonadotrophins, such as equine chorionic gonadotrophins (eCG) and human chorionic gonadotrophins (hCG), increased the rate of ovulation and fertility when used in combination with progestogens in sheep (Vázquez et al., 2004) and (Martinez et al., 2015); however, the pregnancy rate is lower than through their natural estrous cycle. An estrous synchronization system, known as Ovsynch exists for ewes (Martínez et al., 2011). This system combines the gonadotrophins releasing hormone (GnRH) with prostaglandin F2α (PGF2α) to overcome problems and limitations in detecting estrous. With this method, a follicular wave is produced for the increasing GnRH pulses, causing the dominant follicle atresia and a new follicular wave is produced two or three day’s later (Colazo et al., 2004). After a period of seven to nine days, luteolysis is induced by applying PGF2α. A second dosage of GnRH 48 h after PGF2α causes synchronization of ovulation 8 h later. This study was conducted to evaluate the effect of sponges with plus eCG with natural mating, compared to the administration of two doses of GnRH plus Chloprostenol (PGF2α) and natural insemination (hand mating) at fixed time on the pregnancy rate and prolificacy in Rahmani ewes under tropical conditions. Also, provides a complete picture of dynamics of selected biochemical blood parameters in ewes from diestrous to next mid lactation period. This biochemical picture wants to supply the normal range of the most commonly used blood serum parameters during the different reproductive stages.
MATERIAL AND METHODS
Location: The study was conducted between the months of February and July of 2015, at a private farm on middle delta of the Nile, Al Gharbia Governorate. The predominant climate is warm sub-humid and an average temperature of 23ºC.
Animals: The animals used were second lambing ewes (n=30), weighing 45.6 ± 2.7 kg with a minimum of 60 day postpartum. Animals used were in a completely randomized design (2 groups) with 15 ewes for treatment. Each group was housed in 10 × 10 m pens with fixed feeders and watering devices for adding access to water and sufficient shade. The ewes were subjected to semi-intensive management which included sufficient grass from 7 am to 4:00 pm.They were offered 500 gm of concentrate ration (crushed corn, wheat bran, line seed cake and mineral salt) per ewe, in metal troughs. Fifteen days before the trial started, all of the ewes were dewormed orally levanide (levamizole 3% + Oxyclozanide 6%, Egypt) and were given an intramuscular injection (IM) of AD3E vitamins (DevedryMed, Egypt).
Procedures:Drugs of estrous synchronization protocols were MPA according to (Nasra, 2002), eCG (Folligon, Intervet international B.V. Boxmeer-Holland), GnRH (Receptal, Intervet international GmbH-Germany) and PGF2α, Chloprostenol (Estrumate, Schering-plough, Animal Health- Germany). The treated Ewes were grouped as follows: Group1: was considered the control treatment, in which on day 0 a sponge with 65 mg MPA was provided and withdrawn at day 12 later when 200 IU eCG was injected IM, During days 12 to 15, ewes were bred through controlled natural mating. Group2: was given 100 μg GnRH on day 0, and 7.5 mg Chloprostenol on day 5, 100 μg GnRH was administered again on day 7, and fixed time natural insemination (hand mating) 12 to 14 hrs., after second injection of GnRH. The ewes of each group were mounted by three, previously tested for health and fertility rams, in group 1 after withdrawing the devices by 12 hrs. To ensure that they were mounted, and 12 hrs. after second injection of GnRH in group 2.
Blood sampling:The first blood samples were withdrawn once from all ewes, at the same hour (7 am), before pregnancy during the Diestrus phase (5 days after the last estrous). Thereafter, blood was sampled on week 4 of pregnancy (early gestation), on week 20 of pregnancy (late gestation), on week 2 postpartum and on week 6 of lactation (mid of lactation). Blood samples, each of 10 ml, were withdrawn from jugular vein of each animal into vacuum glass tubes containing no anticoagulant. Following standing at room temperature for 20 min, the tubes were centrifuged at 3,000 rpm for 10 min and the serum samples stored at -25°C until analyzed. Part of blood serum was to determine serum concentrations of progesterone by using ELISA Kit “an enzyme immunoassay” for quantitative determination; concentrations below 1 ng/ml were considered indicators of an anestrous stage, and concentrations above 1 ng ml were considered indicators of a reproductive stage Stabenfeldt et al. (1969). Another part of blood serum used for determination of total protein, according to Henery (1968), total lipids according to Zoller and Kirsch (1962), and total cholesterol according to Watson (1960).
Pregnancy rate: The pregnancy rate was determined 50 days after the last mount or fixed time natural insemination (FTNI), with a real-time B mode ecograph and 5-7 MHz trans-abdominal convex transductor.
Prolificacy:was quantified after lambing in the two groups of the treatments by dividing the number of lambs born by the number of ewes that gave births.
Statistical analysis: All results were expressed as means and standard deviations (SD). The analysis of variance (ANOVA) was used to test the overall significance of differences among the means. The data obtained on pregnancy rates and prolificacy was analyzed by the chi-square test according to Snedecore and Cochran (1987).
We found that all ewes in Group1, presented estrous during the first 48 hrs. After removing the sponges. In (group 1): Application of 50 mg MPA, plus 200 IU eCG with controlled mounting; after 24-36 hrs. Interval from estrus onset, eight (53.3%) ewes showed signs of estrous and initiated estrous 100 % with an average time of 37.2 ± 8.43 hrs. after sponge removal. In (group 2): Application of 100 μg GnRH, plus 7.5 mg Chloprostenol, plus 100 μg GnRH and fixed time natural insemination (with controlled mounting); after 12 to 14 hall ewes applied on by fixed time natural insemination with hand mating. There were differences (p>0.05) in pregnancy rates between the treatments of the two groups. The results obtained were 66.66% (n=10) and 53.33% (n=8) for G1 and G2 respectively. There is no difference in prolificacy found among the two treatments (Table 1).
There were differences (p>0.05) in pregnancy rates between the two treatments. The results obtained were 66.66%, (G 1) and 53.33% (G 2) as in table 1. No differences in prolificacy were found among the two treatments (Table 1), Fig. (1).
The values with different lower script letters in the same rows (Tables 3, 4) are significantly different at least (P<0.05).
Serum total protein showed a significant increase during early pregnancy and Diestrous compared to mid lactation. Total lipids level decreased significantly over the mid of lactation versus postpartum and late gestation. Total cholesterol showed significant decreases during postpartum compared to Diestrus.
Estrous induction results in group 1 are similar to those reported by Martínez et al. (2008) and Zarkawi (2011), recorded that, when they applied sponges impregnated with MPA and eCG, estrous began after removing the sponges. They reported a percentage of estrous induction of 90% on Polwarth ewes synchronized with intravaginal sponges with 65 mg MPA plus 250 IU eCG. Also obtained intervals between 36 and 60 h in adult ewes treated with intravaginal sponges impregnated with 65 mg MPA and eCG, while Karaca et al. (2009) reported no differences in estrous response time in ewes synchronized with MPA. Pregnancy rates for G1 were higher than those reported by Fonseca and Torres (2005), who obtained 59.9% using 200 IU eCG. Cueto and Gibbons (2004) reported a pregnancy rate of 47% with MPA, also Alavez et al. (2014) compared the use of a PGF2α analogue (Cloprostenol) IM, with an intravaginal progestagen sponge, pregnancy rates were 53.3 and 60.0 %, respectively. While Catalano et al. (2009) found that, using a higher dosage of eCG (500 IU) plus sponges with MPA, obtained a pregnancy rate of 54% in cross-bred ewes (Frisona x Corriedale). While, Kridli and Al-Khetib (2006) stated that, the use of sponges for estrous synchronization allows controlled lambing in a high percentage of ewes (95.7%) in a short period of time (10 days), with no negative effects in pregnancy rates (86.6%). Also, Dias et al. (2015) found that, DPR “device for sustained progesterone release“ is effective in increasing and maintaining progesterone concentrations, controlling follicular dynamics, promoting synchronized times of ovulation from healthy follicles with pregnancy rates similar to that with use of the CIDR “Controlled Internal Drug Releasing Device (CIDR)”. In addition, Vázquez et al. (2004) mention that injecting 400 IU eCG 48 h before removing the MPA sponges increases ovulation rates and, consequently, pregnancy rates. Powell et al. (2011) reported that, ovulation synchronization with analogues of the hormone GnRH has been used with better results in cattle. The application of GnRH in cattle causes ovulation of the dominant follicle at the moment of treatment, whether in the growth phase of the static phase, causing atresia of the follicles that are not in condition to ovulate and then a new wave of follicle development occurs two or three days after treatment. The ovsynch method has been applied and studied more in cattle than in ovine; Chevel et al. (2003) determined the effects of re-synchronizing after using the ovsynch protocol, administering GnRH (100 μg) on day 21 after AITF in Holstein cows, which reported a pregnancy rate of 70.9 % 21 days later. Stevenson et al. (2006) used the ovsynch protocol in cows plus application of CIDR (1.9 g P4) before the first injection of GnRH. Akif and Kuran (2003) stated that the used of gonadotrophins in ewes stimulates the release of FSH and LH by the anterior hypophysis, which results in an increase in the ovulation rate, and therefore, in the pregnancy rate.
Induction of multiple ovulations is transcendental in the smaller ruminants since higher dividends can be obtained if the ewes produce more than one offspring at a time. Boggio (1997) reported that, the use of GnRH in ovine aims to group estrous in shorter intervals and induce estrous and ovulation in periods in which fertility is reduced because of the season of the year, additional to the time of year or to their nutritional state at the time of treatment (Nazifi et al., 2002) or perhaps because ovulation did not occur in all of the animals after treatment. Nagatani et al. (1998) indicated that different environmental stimuli and nutrition were considered among the most important factors in regulating the reproductive function of the animals. Baumgartner and Pernthaner (1994) found no significant effect of the reproduction stages on the serum concentration of total protein in Karakul sheep. Maternal serum protein concentrations decrease due to an increased fetal growth, and especially the utilization of amino acids from the maternal circulation for protein synthesis in the fetal muscles (Antunovic et al., 2002).
The significant decrease in mid lactation of serum total protein compared to Diestrous and early gestation could be due to a decrease in serum globulin (El-Sherif and Assad, 2001). The lower values of total protein in lactating ewes compared to Diestrous phase prove the high energy need due to milk synthesis which exists in animals, as confirmed by other authors, especially during the early lactation (Bremmer et al., 2000).Significant increase in total lipids in the middle and at the end of pregnancy compared to Diestrous could be ascribed to the higher levels of free fatty acids (FFA) in pregnant than in non-pregnant ewes, caused by increased level of cortisol due to stress induced by pregnancy (Fleming, 1997) as well as increased sensitivity of ewes to epinephrine hormone, which lead to the increase in serum free fatty acids concentrations in late gestation (Revell et al., 2000). The elevated level of total lipids in late gestation compared to Diestrous is probably due to the reduced insulin-mediated inhibition of lipolysis observed in late pregnancy (Schlumbohm et al., 1997). Lipogenesis stimulated by insulin is also responsible for the increased values of total lipids observed in ewes during early lactation. The significant post-partum increase in serum total lipids induces an increased FFA uptake by the liver from circulating plasma, with increased triglyceride storage, as observed in cattle by Grummer (1993). The significant decrease in total cholesterol in late pregnancy has also been reported in other species by Tainturier et al. (1984) in Friesian cows at the end of pregnancy, Bekeová et al. (1987) before parturition in cows and Krajnicáková et al. (2003) in goats. This is probably related to the role of the compound in ovary steroidogenesis so that the total cholesterol concentrations are under control of the complex of factors.
The decrease observed during lactation compared to Diestrous could be ascribed to the increased cholesterol uptake by tissues involved in milk synthesis, because of the normal insulin responsiveness compared to late pregnancy (Nazifi et al., 2002). Total cholesterol concentration in blood serum was increased significantly during pregnancy period this may be due to enhanced progesterone synthesis in the placenta (Sakurai et al., 1997) and it’s decline after parturition due to estrogen which decreased the plasma level (Revell et al., 2000). In conclusion, the implementation of the progestagen-eCG treatment in Rahmani ewes is a good procedure as part of the reproductive management. Applying the (Ovsynch protocol) is a good alternative; more research is required, to prevent low conception rates. Also, biochemical picture should be considered as guidelines for the management strategies for ewes during farming condition. However, further investigations are necessary for the correct interpretation of metabolic diseases in ewes, aimed at avoiding a decline of the productive performance and consequently economic lost.
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