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Estrus and conception are the two most important phases regarding the reproductive health of buffaloes. Estrus detection can increase conception rate, in this regard current research was designed to investigate the effect of methionine with double Ovsynch protocol on reproductive performance of Nili-Ravi buffaloes. The research was performed at Sar Buland Dairy Farm in Faisalabad, Punjab, Pakistan. A total of 40 animals were selected and divided into two groups labeled A and B. Group A was given methionine @25gram per animal per day up to the completion of double Ovsynch protocol of synchronization while Group B was subjected to synchronization protocol only. After completion of the protocol estrus intensity and preovulatory follicular size were monitored then timed artificial insemination (TAI) using frozen thawed semen was performed. Pregnancy rate and early embryonic mortality were checked through ultrasonography at Day 30 and 45 post-AI. Blood samples were taken at day 0 and day of AI then day 30 post-AI to determine serum progesterone and estradiol concentrations. Pregnancy rate, early embryonic death rate, serum estradiol at day 0, and serum progesterone at day 0 and 30 showed statistically non-significant differences between treatment and control groups (P>0.05) while pre-ovulatory follicular size, estrus intensity, and serum estradiol at the day of AI showed a statistically significant difference between treatment and control groups (P<0.05). Concludingly, research illustrates methionine in combination with double Ovsynch protocol improves estrus expression of Nili-Ravi buffaloes.
Abstract
Estrus and conception are the two most important phases regarding the reproductive health of buffaloes. Estrus detection can increase conception rate, in this regard current research was designed to investigate the effect of methionine with double Ovsynch protocol on reproductive performance of Nili-Ravi buffaloes. The research was performed at Sar Buland Dairy Farm in Faisalabad, Punjab, Pakistan. A total of 40 animals were selected and divided into two groups labeled A and B. Group A was given methionine @25gram per animal per day up to the completion of double Ovsynch protocol of synchronization while Group B was subjected to synchronization protocol only. After completion of the protocol estrus intensity and preovulatory follicular size were monitored then timed artificial insemination (TAI) using frozen thawed semen was performed. Pregnancy rate and early embryonic mortality were checked through ultrasonography at Day 30 and 45 post-AI. Blood samples were taken at day 0 and day of AI then day 30 post-AI to determine serum progesterone and estradiol concentrations. Pregnancy rate, early embryonic death rate, serum estradiol at day 0, and serum progesterone at day 0 and 30 showed statistically non-significant differences between treatment and control groups (P>0.05) while pre-ovulatory follicular size, estrus intensity, and serum estradiol at the day of AI showed a statistically significant difference between treatment and control groups (P<0.05). Concludingly, research illustrates methionine in combination with double Ovsynch protocol improves estrus expression of Nili-Ravi buffaloes.
Keywords: Amino acid supplementation; Buffalo; Estrus intensity; Pregnancy; Preovulatory follicular size
Introduction
The tamed river buffalo is frequently referred to as "the living tractor of the East," as used for tillage and transportation. They can consume roughages of poor value, tolerate harsher environments, and become more resilient to several tropical bovine illnesses [1]. On the other hand poor management, climate circumstances, and inadequate feed supplies are key predisposing factors that limit buffalo's productive and reproductive performance [2]. Despite these disadvantages, the use of modern reproductive technologies in buffalo reproduction is very restricted and due to its low breeding potential been ignored for decades.
Early embryonic death and complete absence or silent estrus are the most common issues behind poor fertility with documented frequencies ranging from 21 to 46% [3, 4]. A methionine (amino acid) codon is used to initiate protein synthesis, and it hence plays a vital part in mammalian biological activities [5]. Methionine is converted into S-adenosylmethionine, a product that offers methyl groups for the de novo production of choline, supplementation has also been shown to increase lipid formation in the blastocyst stage, which serves as a source of energy and increases the embryo's survival potential [6]. Substrates and cofactors engaged in methyl methylation are thought to be especially crucial during these critical developmental periods [7]. Methionine strengthens overall animal health, stimulates hepatic lipid metabolism, increases milk protein, and produces an efficient immune response by triggering PMN towards infectious diseases in dairy cows [8]. Methionine decreases the early embryonic death rate and increases estrus intensity which ultimately improves the conception rate through timed artificial insemination.
The double Ovsynch protocol is designed to synchronize ovulation precisely, allowing for timed artificial insemination (TAI) and high conception rates [9]. It is an excellent, well-researched TAI protocol with proven success and significant advantages over most other protocols, addressing challenges in reproductive management such as heat detection, anovular cows, and conception rates [10]. The double Ovsynch protocol also works well as a resynchronization protocol, with higher conception rates compared to regular Ovsynch. Overall, the double Ovsynch protocol offers practical benefits, such as bringing a large percentage of buffaloes into estrus at a predetermined time and improving fertility and conception rates in buffaloes [11]. The obj ectives of this study were to find out the effect of methionine supplementation with double Ovsynch protocol of synchronization on estrus intensity, preovulatory follicular size, serum estradiol, serum progesterone concentrations, early embryonic death rate and pregnancy rate in Nili-Ravi buffaloes. Materials and Methods Field research was conducted in Faisalabad, a city located in the northeastern region of the Punjab province, Pakistan (Latitude: 31.4504° N, Longitude: 73.1350° E) at Sar Boland Dairy Farm. The duration of the research was about two months and forty Nili-Ravi Buffaloes having BCS >3 and age between three to five years were selected and these animals were multiparous with 12 to 15 litters lactation yield. Animals were kept in uniform managemental feeding conditions, the shed was divided into two separate sections, with animals of the experimental group kept in one section and animals of the control group in another section. There was one water manger to ensure a fresh supply of water round the clock. The animals were free in each section having free access to feed and water.
The diet of experimental animals in group A (n=20) was supplemented with Rumen Protected Protein (Menomet(r))
@25g/animal/day up to the completion of double Ovsynch protocol of synchronization. Animals of group B (n=20) were fed with a control diet without Menomet(r).
Blood samples were collected from both groups before injecting first injection of protocol and methionine supplementation, the samples were again collected from all the animals at the days of estrus, at day 30 after TAI for a comparative analysis between control and the experimental group. The buffaloes were restrained individually in the crush before collection and about 10ml of the blood was collected from each animal using a sterile syringe immediately shifted to falcon tube containing lOmg of EDTA (Ethylenediamine tetra acetic acid) as an anticoagulant. Then sent to the private lab for serum progesterone and estrogen concentration evaluation. GnRH analogues containing Buserelin Acetate (0.0042ug/ml) were administered at days 0, 9, 16 and 25 and analogue of PGF201 containing Cloprostenol (250ug/ml) were administered at day 7 and 23 according to double Ovsynch protocol of synchronization [12]. Estrus intensity was monitored and then scored according to Layek et al. [13]. Timed artificial insemination was performed after 12 hours of the last protocol injection. At the time of insemination, all animals were evaluated through ultrasonography for the assessment of pre-ovulatory follicular size. After that artificial insemination (AI) using frozen thawed semen is performed. Then after 30 days of AI ultrasonography to confirm conception and then at 45 days to rule out early embryonic death performed. For evaluation of their statistical significance estrus intensity score, size of pre-ovulatory follicle, serum estradiol (pg/ml), and serum progesterone (ng/ml) were analyzed using T-test between treatment and control groups. Chi-square was used for the determination of the statistical significance of pregnancy rate and early embryonic death rate between treatment and control groups. Results and Discussion The research was designed to investigate the efficiency of double Ovsynch protocol with feeding of methionine on estrus intensity, pre-ovulatory follicular size, pregnancy rate, early embryonic death rate along with serum estradiol (E2) and progesterone (P4) concentrations in Nili-Ravi Buffaloes.
The treatment group in this research showed higher estrus intensity, preovulatory follicular size (POFs), and serum estradiol concentration on day of AI from control group and their difference were statistically significant (P-value <0.05). However, pregnancy rate, early embryonic death rate, serum estradiol, and progesterone concentration at day 0 and serum progesterone at day 30 post AI showed non-significant differences statistically (P-value>0.05) as shown in (Tables 1-3).
Previously there was a non-significant difference of estrus intensity and preovulatory follicular size between treatment and control group after using double Ovsynch protocol of synchronization in treatment group in normal cyclic cows [14, 15]. While other studies supported current research that Ovsynch and double Ovsynch protocols increased follicle diameter in Egyptian buffaloes and Holstein cows [16] where the growth pattern was in line with the research of Sellars et ah, (2006) where double Ovsynch protocol was used in Holstein cows [17].
Another study found that the conception rates of buffaloes having small (9 to < 12 mm), medium (>12 to <14 mm), and large (>14 to 16 mm) POFs at estrus were 9.1%, 70.0%, and 85.7%, respectively [18]. Additionally, a study investigated the effects of POF size on estradiol concentrations, CL diameter, progesterone concentrations, and subsequent pregnancy rate in Murrah buffalo cows [19]. These findings suggest that POFs are an important factor in reproductive outcomes in buffaloes, and larger POFs may lead to improved conception rates and larger corpus luteum.
In the current investigation level of estradiol was almost at the same level in both the treatment and control group at the onset of study but were high at the day of estrus because the follicles in the treated group were larger in size as compare to control group. This variation in estradiol concentration on day of estrus is due to the impact of rumen undegradable protein on growth of follicles especially methionine that increases the maturation of oocytes as in line with the work of An et al., [20] that shows higher the size of foilicle higher will be the estradiol concentration. Another study added the Ovsynch protocol does affect estradiol levels in buffaloes. According to the research [21], the Ovsynch protocol reduces the incidence of anestrus and improves fertility by suppressing prolactin secretion, which in turn leads to enhanced gonadal hormone production including estradiol-17p. The concentration of estradiol in the current study was also in line with the research conducted by Bello et al. [22] there concentration of estrogen showed same pattern. These findings indicate that the Ovsynch protocol affects estradiol levels positively, contributing to the success of synchronized estrus and subsequent fertilization efforts in buffaloes.
Progesterone was higher at Day 30 in the experimental animals but the nonsignificant difference from control group compared to previous study by Richardson et al. [23] which have shown nonsignificant differences of progesterone concentration. The mean concentration of progesterone was 1.56±0.21 in the treated group in the current study related to research ofPrajapatie^ al. [24] and Kutzler et al. [25] shows the same increasing pattern of progesterone concentration after double Ovsynch. It is evident that the Ovsynch protocol has an impact on progesterone levels in buffaloes but current research did not return specific results regarding the direct effect of methionine supplementation on progesterone levels in buffaloes.
Pregnancy rate by insemination at proper time is a way to get better pregnancy results but limitations occur in the timed artificial insemination because the time of ovulation and estrus behavior varies in animals. But fixed-time Al increases the pregnancy rates per Al and decreases the pregnancy losses [26]. This research showed the pregnancy rate was high in the treated group as compare to control but there was statistically non-significant difference (P-value>0.05) between them similar to the previous study of Khalifa et al., (2011) where Ovsynch protocol was used [27]. Ovsynch protocol generally improves pregnancy rates in buffaloes, particularly in cases of anestrus or suboptimal breeding seasons. For example, a study comparing the Ovsynch and progesterone-based protocols showed that the conception rate was significantly higher with the progesterone-based protocol than with the Ovsynch protocol (66.7% vs. 30%) in subestrous buffalo during the low-breeding season [28]. While discussing the Early Embryonic Death rate current research showed less number of deaths in treated group as compare to control group but there differenece were statistically nonsignificant and there is limited research on this topic but role of methionine addition have been studied inHolsteinFreisian cows diets illustrated the improvement in survival rate of preimplantation embryos, and embryonic death dropped from 19% to 6% in cows fed methionine [29] it was found that cows fed methionine had more lipid droplets inside the preimplantation embryo, which could be used as energy by the embryos. The study also showed that methionine is the first limiting amino acid for dairy cattle, and the lack of methionine limits cows in producing protein in the milk. However, there is no direct evidence of the impact of methionine supplementation on early embryonic death rates. Another study showed that buffaloes treated with GnRH at day 20 of Al had lower early embryonic mortality compared to those without GnRH treatment, especially in aged, multiparous, low body condition score (BCS), and low milk-producing buffaloes [30]. These findings suggest that supplementing methionine with the double Ovsynch protocol may improve early embryonic survival rates in buffaloes, but further research is needed to confirm this hypothesis. The role of methionine combined with double Ovsynch protocol has not studied yet so further research is needed to fully understand the combined effects of methionine supplementation with double Ovsynch protocol on estrus intensity, preovulatory follicular size, pregnancy rate, early embryonic death rate, serum P4 and E2 concentrations in different populations of cattle and buffaloes. Concludingly, methionine supplementation with double Ovsynch protocol of synchronization helps in the improvement of estrus expression in Nili-Ravi buffaloes as they have the role in better presynchrony of growing follicles and increase their size which ultimately leads to formation of larger corpus luteum. Authors' contributions Conducted the experiments: M Waqas, Conducted making design: H Jamil, MM Hassan, W Hasan & MS Tariq, Collected and analyzed the data: A Munawar & Y Asghar.
Acknowledgment
We gratefully acknowledge the contributions and support of various individuals and organizations that made this research possible. Special thanks are to the farm manager of Sar Bui and dairy farm for his assistance in data collection, analysis, and interpretation. We also extend our appreciation to Ch. Basharat for financial support. Furthermore, we thank the Chairman of the Department of Theriogenology, University of Agriculture Faisalabad for providing the necessary facilities for conducting this study. Lastly, we express our gratitude to the farmers and personnel involved in the care and management of Nili Ravi buffaloes during this research.
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