Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 11 (november 2021) : 1279-1285

An Alternative Buffalo Urine-based Non-invasive Early Estrus Test using Wheat and Mung Bean Seed Germination

Tanvi Bhatia, Varij Nayan, Rakshita Singh1, Chhama Singh1, Anuradha Bhardwaj2, Sunil Kumar1, M. Naveen Swaroop1, S.K. Onteru3, R.K. Sharma1, Anurag Bharadwaj1, Dheer Singh3, A.K. Mohanty3
1Division of Animal Physiology and Reproduction, Molecular Endocrinology, Functional Genomics and Computational Biology Laboratory, ICAR-Central Institute for Research on Buffaloes, Hisar-125 001, Haryana, India.
2Animal Biotechnology Laboratory, ICAR-National Research Centre on Equines, Hisar-125 001, Haryana, India.
3ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India.
Cite article:- Bhatia Tanvi, Nayan Varij, Singh Rakshita, Singh Chhama, Bhardwaj Anuradha, Kumar Sunil, Swaroop Naveen M., Onteru S.K., Sharma R.K., Bharadwaj Anurag, Singh Dheer, Mohanty A.K. (2021). An Alternative Buffalo Urine-based Non-invasive Early Estrus Test using Wheat and Mung Bean Seed Germination . Indian Journal of Animal Research. 55(11): 1279-1285. doi: 10.18805/IJAR.B-4237.

ABSTRACT

Background: The silent estrus problem in buffaloes is one of the major bottlenecks in buffalo management. Here, we present for the first time a simple and urine-based non-invasive seed germination inhibition test to detect the early estrus in buffaloes.

Methods: The urine samples were collected from buffaloes on 0, 6, 10, 14, 18, 21 and 28 days after detection of signs of heat. The mungbean and wheat seeds (n=15 each) were treated with diluted (1:2 and 1:4) and undiluted urine samples. The results were analyzed in terms of germination inhibition percentage (GI%) and shoot length (SL) measurements. Control was established using distilled water in place of urine.

Conclusion: In 1:4 dilution urine samples, a significant (P ≤ 0.05) decrease in GI% and SL were observed after 48 hours and 5 days, respectively in both the seeds, which could be useful as an estrus test. No significant results were observed for GI% and SL with undiluted and 1:2 diluted samples in both seeds. Our findings demonstrate that GI% and SL were inhibited around peri-estrus events, increased from 10th-14th days and then gradually decreased with 1:4 dilution of urine. This estrus test may assist the farmers in timing AI.

INTRODUCTION

The dairy sector occupies one of the most important positions among all livestock sub-sectors of our agricultural economy in India. Buffaloes are one of the main pillars of the dairy economy in India. However, several factors affect the productivity of buffaloes. The follicular microenvironment (Fortune et al., 2004), including the IGF system and PAPP-A (Nayan et al., 2013) as well as the reproductive hormones such as FSH, LH and inhibin (Bhardwaj et al., 2012; Bhardwaj et al., 2013) together with the epigenetics-nutriment-nurture crosstalk (Nayan et al., 2015) provide greater roles in the reproductive outcome of the dairy animals. Another very important factor is the failure of early estrus or heat detection in buffalo breeding and it is recognized as a critical issue for the farmers in dairy entrepreneurship (Hegde, 2019). Estrus detection followed by the successful impregnation of buffaloes is the most important aspect on the dairy farm (Rao et al., 2013). With limited availability of pregnant mare serum gonadotropin (PMSG), research was also made towards production of recombinant equine chorionic gonadotropins (Bhardwaj et al., 2019; Bhardwaj et al., 2020) and they may have implications in buffalo estrus induction and fertility. The estrous cycle comprises of the proestrus, estrus, metestrus and diestrus phases. Estrus detection is the intended act to observe and trace the estrus so that the artificial insemination (AI) of the animal takes place at this time. This is the period in the estrous cycle when the female is fertile and can conceive if mated (Senger, 2003).
       
However, the silent heat in buffaloes is one of the major bottlenecks (Jainudeen, 1988). The incidence of silent estrus may be higher in herds using AI in comparison to natural mating and this may indicate that the problem often lies with estrus detection instead of the animal itself (Suthar and Dhami, 2010). Overcoming this critical barrier to buffalo reproduction will certainly improve the production efficiency of buffaloes. Estrus symptoms in buffaloes are less evident than those in cattle, such as less than a third of buffaloes in estrus can be detected by homosexual behavior. Other heat symptoms like swollen vulva, mucous discharge from the vagina and increased urination frequency are very feeble. Estrus in buffaloes can also be detected by their reactions to a vasectomized male/ teaser or an androgenized female buffalo (Rajanarayanan and Archunan, 2004). Besides these visual signs, other ways to detect estrus include automated systems like pedometry or heat watch, chin ball markers, Kamar estrus mount detectors and ELISA tests detecting progesterone. All these tests either involve the role of experts or exceed the budget of poor farmers. It is therefore very necessary to evolve a simple test that can detect estrus in buffaloes as early as possible and without missing any silent heat.
 
Recently, several newer approaches are being tried based on salivary fern pattern (Ravinder et al., 2016), direct saliva transcript analysis for Hsp70 and TLR4 (Onteru et al., 2016). Some newer approaches to capture the ovulation event and timing AI include tests as a dipstick or lateral flow assay based on the functionalization of gold nanoparticles for the detection of buffalo luteinizing hormone (Nayan, 2015; Nayan et al., 2020). Here, we have adapted the ancient Egyptian knowledge of a simple non-invasive bioassay called Punyakoti test or seed germination inhibition test to detect pregnancy in cattle and buffaloes (Veena et al., 1997; Krishna and Veena, 2009) for use as an estrus test in buffaloes. Working on this approach, a modified seed germination inhibition test was deployed to foretell early estrus for the first time in Murrah buffaloes.

MATERIALS AND METHODS

Sample collection
 
Collection of buffaloes urine samples (n=2) was made at the animal farm of ICAR-Central Institute for Research on Buffaloes, Hisar (Coordinates: 29°10'49.40" N, 75°42'24.87" E) during normal estrous cycle on days 0, 6, 10, 14, 18, 21 and 28 after the detection of signs of heat (Fig 1). The samples were collected during natural micturition in clean, sterile plastic bottles, filtered and immediately transferred to the laboratory at 4°C on ice. The samples were collected from Murrah buffaloes aged between 4 to 5 years in August and September months in the year 2019 during the average climatic temperature of 31°C and average humidity of 75%.
 

Fig 1: The visible signs of estrus in female buffalo.


 
Test procedure
 
Two different seed types, viz. wheat and mung bean seeds were selected for the study. Fifteen seeds each of wheat and mung bean were placed on the blotting paper in sterile Petri dishes for analysis and two dilutions of a urine sample with distilled water, i.e. 1:2 and 1:4, as well as an undiluted urine sample, were added. The control test was also carried out with the addition of only water to the seeds. Samples were covered and kept at 37°C. After five days, the seeds were examined for germination rate and shoot length (SL). SL of seeds was measured with the ruler. Germination inhibition (GI) percentage (%) and SL of control and test groups were calculated and compared (Bethapudi et al., 2015). GI% was calculated by the following formula:
 
 
Germination inhibition percentage
 
It was defined as the number of seeds not germinated out of the total number of seeds taken in the experiment.

Statistical analysis
 
GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, California, USA, www.graphpad.com) and Microsoft Office Excel 2007 were used to calculate the standard deviation (SD) between mean values of SL and GI% by the post hoc analysis. A value of P≤0.05 was considered significant.

RESULTS AND DISCUSSION

Germination inhibition percentage (GI%) and shoot length
 
After the wheat and mung bean seeds were plated in the urine sample of buffaloes (number 4917 and 4625), the measurements were made for the germination inhibition percentage (GI%) after 48 hours and the average shoot length (SL) after 5 days. The data and photographs are depicted in Fig 2-6. The results indicated that with 1:4 dilution of urine, a significant (P£0.05) decrease in GI% was observed after 48 hours as compared to the control plate (with distilled water only). In 1:4 dilution of urine, the GI% decreased till 10th day of the estrous cycle and after that, it suddenly increased till the 18th day followed by a gradual decrease afterward till the 28th day. The germination inhibition was 100% in undiluted urine samples, whereas it varied regarding the days of the estrous cycle in 1:2 dilution and 1:4 dilution of urine samples. The variations observed in the trend may be due to some environmental factors or the physiological condition of an animal during sample collection. Similarly, the average shoot length (SL) growth of both the seeds in 1:4 diluted urine of both animals showed a gradual increase up to the 14th day of the estrous cycle and after that, it suddenly decreased on the 18th day and again gradually increased till the 28th day. A comparison of the seed lengths of both types of seeds in 1:4 dilution of urine though heatmap showed vast variation between 14th and 18th day of the estrous cycle (Fig 7). The pattern of changing color was similar before and after the 18th day but on the 18th day, germination inhibition was 100% and shoot germination was less. Similar results were represented by the culture plate of both seeds with 1:4 dilution of urine sample in which no germination was visible on the 18th day (Fig 6). The qualitative and quantitative differences in seed reserves may relate to the germination characteristics of seeds. In one of the studies, it was reported that the protein, fat and starch reserves in dry seeds were not significantly correlated with germination rate and germination percentage, but soluble sugar and soluble protein concentration at every germination stage were positively correlated with germination rate. Soluble starch was primarily consumed during seed inhibitions while soluble protein consumed especially after imbibition to the highest germination stage. Still, it had not much difference observed in fat content during seed germination (Zhao et al., 2018). Undiluted urine was inhibiting seed germination and with 1:2 dilution also seed germination was minimal. This inhibition may be because of abiotic stress caused due to high salt concentration or some metabolites. Earlier, it was also enumerated in cases of pregnancy that the inhibitory effect of urine may be due to the presence of abscisic acid (ABA), a plant hormone that causes seed dormancy in nature (Dilrukshi and Perera, 2009; Skálová et al., 2017). Generally, several metabolic processes including the synthesis of the hydrolytic enzymes are activated on seed imbibition. It then causes the reserve food material hydrolysis into simpler forms required for the embryo growth. The abiotic stresses can potentially influence the germination of the seed, seedling establishment and development through the intervention of various factors, viz. reduced water availability, hormonal imbalance, altered mobilization of stored reserves and alterations in the soluble protein structural organization (Ali and Elozeiri, 2017).
 

Fig 2: A) Germination Inhibition per cent (GI%) and B) Average SL of wheat seeds in urine sample of buffalo No. 4917 in estrous cycle. After 48 hours, the GI% of wheat seeds in urine sample was more as compared to control plates. There was 100% germination inhibition in undiluted urine sample whereas in 1:2 dilution and 1:4 dilution GI% varied with respect to the days of estrous cycle. In 1:4 dilution, the GI% decreased till day 10 of estrous cycle after that it suddenly increased till 18th day followed by gradual decrease afterwards till 21st day. Similarly, the average SL in 1:4 dilution increased till 14th day of estrous cycle after that it suddenly decreased on 18th day and again gradually increased till 28th day.


 

Fig 3: A) Germination Inhibition per cent (GI%) and B) Average SL of mungbean seeds in urine sample of Animal No. 4917. After 48 hours, the GI% of mungbean seeds in urine sample was more as compared to control plates. There was 100% germination inhibition in undiluted urine sample whereas in 1:2 dilution and 1:4 dilution GI% varied with respect to the days of estrous cycle. In 1:4 dilution, the GI% decreased till day 10 of estrous cycle after that it suddenly increased till 18th day followed by gradual decrease afterwards till 28th day. Similarly, the average SL in 1:4 dilution suddenly decreased on 14th day and slightly increased on 18th day and again gradually increased till 28th day.


 

Fig 4: A) Germination Inhibition per cent (GI%) and B) average SL of wheat seeds in urine sample of Animal No. 4625. After 48 hours, the GI% of wheat seeds in urine sample was more as compared to control plates. There was 100% germination inhibition in undiluted urine sample whereas in 1:2 dilution and 1:4 dilution GI% varied with respect to the days of estrous cycle. In 1:4 dilution, the GI% decreased till day 10 of estrous cycle after that it suddenly increased till 18th day followed by gradual decrease afterwards till 28th day. Similarly, the average SL in 1:4 dilution suddenly decreased on 14th day and slightly increased on 18th day and again gradually increased till 28th day.


 

Fig 5: A) Germination Inhibition per cent (GI%) and B) average SL of mungbean seeds in urine sample of Animal No. 4625. After 48 hours, the GI% of mungbean seeds in urine sample was more as compared to control plates. There was 100% germination inhibition in undiluted urine sample whereas in 1:2 dilution and 1:4 dilution GI% varied with respect to the days of estrous cycle. In 1:4 dilution, the GI% decreased till day 10 of estrous cycle after that it suddenly increased till 18th day followed by gradual decrease afterwards till 28th day. Similarly, the average SL in 1:4 dilution suddenly decreased on 14th day and slightly increased on 18th day and again gradually increased till 28th day.


 

Fig 6: Seed germination inhibition test of urine samples of 1:4 dilution in animal no. 4917 with (A) Wheat (B) Mungbean and in animal No. 4625 with (C) Wheat and (D) Mungbean. ‘C’ indicated the control plate with distilled water and 0 day, 6th day, 10th day, 14th day, 18th day, 21st day, 28th day indicate experimental plate with 1:4 dilution urine samples. Seed germination was gradually increased up to 10th or 14th day and suddenly inhibited on 18th day and then starts gradually increasing with both seeds in both animals.


 

Fig 7: Heatmap represents the mean shoot length of Wheat and Mungbean in both buffaloes (number 4917 and 4625) urine samples of 1:4 dilution. In this, ‘W’ and ‘M’ with the animal number indicates the type of seed used with that animal urine sample in the experimental plate. Yellow to blue color range indicates the growth of SL from lowest to highest. The SL was gradually increased up to 14th and it suddenly decreased on the 18th day and again gradually increased after the 18th day.

CONCLUSION

Early detection of estrus has become an important requirement in dairy farming and animal breeding. In the results, the trend in seed germination of wheat seeds in 1:4 dilution of urine showing nearly no seed germination just after the gradually increased seed germination trend and again follow the same pattern of the result after 18th day. Therefore, it can be easily concluded that after 10 days again animal comes in a cycle and ready for AI. Therefore, we can predict the sequence of events preceding estrus and prepare the animal for future events by formulating this small experiment. Undiluted urine and urine in 1:2 dilution was inhibiting seed germination because of abiotic stress. It seems that in different germination stages, the soluble protein and starch content of both seeds considerably decrease. The salinity of urine or some uncharacterized metabolite resulted in lower seed imbibition that led to lesser protein mobilization in wheat and mung bean seeds. However, the results still need validation in a large sample size with an exploration of the factors, metabolites and other substances responsible. This test has the potential to form an easy early estrus detection test for animals that can help the farmers.

ACKNOWLEDGEMENT

The authors are very grateful to Director, ICAR-Central Institute for Research on Buffaloes-Hisar, for providing the necessary facilities for this work. The project funding under the CIRB Institute project (IXX12688) and the Bill and Melinda Gates Foundation (OXX04350) is duly acknowledged. Facilities were also availed from CABin Project (OXX04610) endorsed to ICAR-CIRB.

CONFLICT OF INTEREST

The authors declared that they have no conflict of interest related to this research.

AUTHORS' CONTRIBUTION

All authors contributed in designing, experimentation, analysis and manuscript preparation and finalization. All the authors finally approved for publication of the article.

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