The study was conducted in 94 cyclic Murrah buffaloes with aim to develop a short-term estrus synchronization protocol consisting of Estradiol benzoate after PGF2α and evaluation of estrus response, luteal profile and conception rae. The buffaloes were divided into two groups with Group-I (n = 40) received PGF2α day 0 and AI was performed twice at 72 and 84h of PGF2α injection while, in Group-II (n = 54): Estradiol benzoate (1mg) 24h of PGF2α injection and AI was performed twice at 72 and 84h of PGF2α injection. Blood sampling for hormonal estimation and USG was carried out before the treatment (day 0), day 1, at the time of first-AI, and day 5 and 12 post-AI. The buffaloes showing intense estrus response were significantly (P < 0.05) greater in Group-II (33.33%; 18/54) than Group-I (7.50%; 3/40). FSCR (%) was higher in Group-II (33.33%; P > 0.05) than Group-I (27.5%). Plasma estrogen concentration of Group-II was significantly (P < 0.05) higher than Group-I on day of AI. The plasma estrogen, IGF-1 concentration was greater (P < 0.05) in pregnant than their non-pregnant counterparts in both groups. The plasma P4 (ng/ml) concentration of pregnant buffaloes on day 5 and 12 post-AI was significantly (P < 0.05) greater than non-pregnant buffalo in both groups. So, it was concluded that short term estrus synchronization protocol with administration of Estradiol benzoate 24h after PGF2α improved the estrus response, plasma estrogen concentration at AI with better post-ovulation luteal profile in cyclic Murrah buffaloes.
Research Article
Impact of PGF2α and Estrogen based short term estrus synchronization protocols on estrus expression, luteal profile and conception rate in cyclic Murrah buffaloes
https://doi.org/10.21203/rs.3.rs-2755643/v1
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Buffalo
Estradiol benzoate
Murrah
PGF2α
Short-term protocol
The importance of buffaloes in Indian dairy industry can be reflected from the fact that despite their lesser numbers than cattle (109.85 versus 192.49 Million; 20th Livestock Census India, 2019), the contribution in total milk production is around 50% from buffaloes (DAHD Annual report, 2019-20). But, poor reproductive efficiency of buffaloes due to delayed puberty, silent estrus, summer anestrus, prolonged post-partum anestrus and ultimately longer inter-calving period (Madan and Prakash 2007) is a major problem. Also, the variability in the duration of estrus from 4–64 h (Baruselli et al. 2001) and poor estrus detection (Purohit and Rao 2018) creates a hurdle in predicting the timing of ovulation thus, limiting the success of artificial insemination (AI) program. The reproductive events like ovulation, is controlled by interplay of several hormones, growth factors and metabolites in the animal body which can affect the ovulation timing (Mirmahmoudi and Parkash 2012).
To counter the problem of poor estrus expression and unpredictable ovulation timing different fixed time AI (FTAI) protocols consisting of various hormonal combinations have been investigated (Baruselli et al. 2001; Paul and Parkash 2005; Cirit et al. 2008; Mohan et al. 2009; Mirmahmoudi and Parkash 2012; Mirmahmoudi et al. 2014; Singh et al. 2022; Saini et al. 2023). In literature, hormonal protocols using progesterone (P4), estradiol, prostaglandin, gonadotropin releasing hormone (GnRH) and equine chorionic gonadotropin (eCG) have been reported to improve the pregnancy rates in buffaloes (Dhaka et al. 2019; Sharma et al. 2021). It has been inferred that all of the existing FTAI protocols are based on the abolishment of the existing follicular wave and start of the new wave, as well as the longer duration with higher implementation cost and multiple administration of injections makes the use of these protocols limited in the field conditions. The use of FTAI programs like Ovsynch and Heatsynch have resulted in the successful pregnancy rates of 30–40% in field conditions (Paul and Parkash 2005; Mohan et al. 2009).
Therefore, it is important to develop some short term estrus synchronization protocols with practicability and comparable results to the existing standard protocols. Studies have reported acceptable pregnancy outcomes by modifying existing follicular wave through use of short-term estrus synchronization protocols in cattle (Cirit et al. 2008; McArt et al. 2010; Jeong et al. 2013; Bandai et al. 2020) and buffaloes (Atabay et al. 2020). These studies used different combinations of PGF2α, GnRH and estradiol benzoate. Recently, Atabay et al. (2020) reported successful use of short term fixed-AI protocol in buffaloes by using different combination of PGF2α and GnRH. Additionally, results of studies in cattle had concluded that combination of PGF2α and estradiol benzoate has greater potential to improve pregnancy rates after FTAI as compared to combination of PGF2α and GnRH (Cirit et al. 2008; Bandai et al. 2020).
The use of estradiol instead of GnRH reduces the timing of ovulation after PGF2α treatment by about 4h as well as results in marginal increase of pregnancy rates (Cirit et al. 2008). The advantage over effect of Estradiol over GnRH in terms of better estrus response, greater uterine tonicity, ease of insemination with reported good ovulation and pregnancy rates in heatsynch (Mohan et al. 2009; 2010) and estradoublesynch (Mirmahmoudi et al. 2014) protocols in buffaloes makes it pertinent to use estradiol instead of GnRH. Also, in terms of economics the use of estradiol is a cheaper affair as compared to GnRH.
But, there is lack of literature pertaining to use of short-term estrus synchronization protocols in buffaloes by including combination of PGF2α and estradiol benzoate. Therefore, it was hypothesized that combinations of PGF2α and Estradiol benzoate (EB) could modify the exiting follicular wave with increased circulatory Estradiol concentration, better estrus response and resultant improved pregnancy rate in the normal cyclic buffalo. The study was planned with the objectives 1) to study the estrus behaviour response with or without administration of Estradiol benzoate after PGF2α and, 2) to compare the endocrine profile (progesterone, Estradiol and IGF-1), post-ovulatory luteal profile and pregnancy rate using short term estrus synchronization protocols with or without use of estradiol benzoate after PGF2α in normal cyclic buffaloes.
The current study was conducted in buffalo at the Government Livestock Farm, Sirsa Road, Hisar and nearby areas after prior approval from Institutional Animal Ethics Committee (Registration: 1669/GO/ReBiBt-S/Re-L/12/CPCSEA), COVS, LUVAS (Hisar) vide approval number- VCC/IAEC/2022/624 − 51; dated- 10-05-2022. The distribution of animals in each group was done randomly to avoid any inter-farm differences under each treatment group. The study was conducted on a total of ninty four (n = 94) normal cycling buffaloes (> 90 days post-partum) between 2nd and 6th parity with 3–4 body condition score out of 5 scale. .
Also, only those buffaloes were considered which had surpassed 90 days post-partum with no manifestation of estrus signs. The buffaloes having history of any pathology of the reproductive organs and aberrant cervico-vaginal mucus discharge, were not part of this study. The feeding practices followed were according to the availability of seasonal green fodder with required concentrates as per milk yield of the individual animal. The animals were provided with 1% mineral mixture and free access to drinking water. The buffaloes were examined twice at 11 days apart through trans-rectal ultrasonography (USG) to ascertain the presence of CL. Furthermore, the blood was also collected on both days of examination to confirm the cyclicity through plasma progesterone concentration. The buffalo had plasma progesterone concentrations > 1 ng/ml on either of the sample was considered as cyclic.
Experimental Design
The buffaloes under the study were randomly distributed into two treatment groups. These buffaloes in respective groups were subjected to administration of single PGF2α and PGF2α followed by EB as categorized below:
Group-I (n = 40)
The buffaloes in this group (n = 40) received a single dose of injection PGF2α (Cloprostenol sodium, 500µg, Zolcol®, Carus Pharmaceuticals Ltd. India) intramuscular (IM) on day 0 and fixed time artificial insemination (FTAI) was performed twice at 72 and 84h of PGF2α injection. The schematic presentation of treatment protocol is described in Fig. 1. Blood sampling for hormonal estimation and USG was carried out before the treatment (day 0), day 1, at the time of first-AI, and day 5 and 12 post-AI.
Group-II (n = 54)
The buffaloes of this group (n = 54) received a single dose of PGF2α (Cloprostenol sodium, 500µg, Zolcol®, Carus Pharmaceuticals Ltd. India) IM on day 0 followed by injection Estradiol Benzoate (1mg, PregHeat®, Virbac Pharmaceuticals India Ltd.) 24h after the injection of PGF2α IM and FTAI was performed twice at 72 and 84h of PGF2α treatment (48 and 60h after injection of Estradiol benzoate; EB). The schematic presentation of treatment protocol is described in Fig. 1. Blood sampling for hormonal estimation and USG was carried out before the treatment (day 0), day 1, at the time of first-AI, and day 5 and 12 post-AI.
Estrus response
The estrus signs showed by the individual animal were recorded during morning and evening hours for confirmation of estrus. The most common estrus signs recorded were frequent micturition, vulvar swelling, mucus discharge, restlessness, bellowing, standing heat, uterine tone, sniffing/licking, aggression and mounting on others (if observed). The buffalo showing various intensities of estrus response were recorded individually. Doka/TET (Temporary engorgement of teats) before the actual onset of estrus was also noted for each animal. Intensity of estrus was described on the basis of total score. Total score for each animal observed was classified into three categories such as weak (1–11), moderate (12–22) and intense (23–33) as described in Table 1 (Layek et al. 2011). The estrus induction rate (%) was calculated for each estrus synchronization protocol as percentage of buffaloes which exhibited the signs of estrus out of all buffaloes subjected to estrus synchronization protocol.
| Estrus Symptom | Frequency of expression during an estrus period | ||
|---|---|---|---|
| Weak (Score 1) | Moderate (Score 2) | Intense (Score 3) | |
| Standing heat | < 9 | 9–14 | > 14 |
| Mounting on others | < 9 | 9–14 | > 14 |
| Frequent micturition | < 3 | 3–4 | > 4 |
| Vulvar swelling | + | ++ | +++ |
| Mucus discharge | Absent | Slight | Copious |
| Restlessness | Mild | Moderate | Pronounced |
| Bellowing | Very seldom | Occasional | Often |
| Uterine tone | + | ++ | +++ |
| Licking/Sniffing | < 6 | 6–8 | > 8 |
| Doka/TET | + | ++ | +++ |
| Aggression | Mild | Moderate | Pronounced |
Ultrasonography (USG)
The trans-rectal ultrasonography for a subset of 15 buffaloes from each group was performed using ultrasound machine (SonoScape®; SonoScape Medical Corp.) before the treatment (day 0), on day 1, at the time of first-AI, and day 5 and 12 post-AI. The follicular and luteal structures of each ovary with their dimensions were recorded for each buffalo subjected to USG. The diameter of largest follicle (LF) on day 0 and day 1, Pre-ovulatory follicle (POF) diameter on day of AI and area of corpus luteum (CL) on day 0 was recorded. The diameter of the follicles/CL was determined by measurement of the largest and widest diameter of the follicles/CL and thereafter, average diameter calculated (Pierson and Ginther, 1985). Additionally, the CL area was recorded on day 5 and 12 post-AI also. The CL area was calculated using formula π X (diameter of CL/2)2. Ultrasonography was performed at day 45 post-AI for pregnancy diagnosis.
Blood sampling and hormone analysis
Blood sampling for hormonal estimation was carried out before the treatment (day 0), day 1, at the time of first-AI and on day 5 and 12 post-AI. Blood samples were collected from jugular vein of buffalo (n = 15 of each group) in 10 ml vacutainer vial containing sodium heparin as anticoagulant. Immediately after blood collection, samples were transferred to the laboratory for separation of plasma. Plasma separation was performed by using centrifuge machine at 1500xg for 15 minutes. Aliquots of plasma were taken in eppendorf tube and stored at -20°C until estimation of hormones.
Plasma progesterone (P4) and estradiol-17β (E2) concentrations were estimated by using a commercially available Direct Immunoenzymatic Assay Kit (CALBIOTECH, USA) while, Plasma insulin like factor-1 (IGF-1) concentration was estimated by using commercially available Sandwich ELISA kit (BT-LAB, Korain biotech).
Statistical analysis
Statistical software IBM SPSS Statistics 24.0 (SPSS Inc., Chicago, IL, USA) was used for making the statistical comparison of the data. The data obtained from both the groups were analysed using one-way ANOVA for comparison of different hormonal concentrations and USG data of all days within each group. Chi-square test was used for comparison of percentage data (FSCR and estrus response) of all groups. Unpaired Student’s T-test was used for comparison of data between pregnant and non-pregnant animals of each group. The values were defined significant at a P-value ≤ 0.05. The values were expressed as mean ± standard error or percentage.
Estrus response
The percentage of buffaloes showing intense estrus response (7.50%; 3/40) in Group-I was significantly (P < 0.05) lesser than those showing weak (52.50%; 21/40) and moderate (40.00%; 16/40) estrus response however, percentage of buffaloes exhibited weak and moderate estrus response were similar (P > 0.05; Table 2). In Group-II, the percentage of buffaloes showing weak estrus response (12.96%; 7/54) was significantly (P < 0.05) lesser than moderate (53.71%; 29/54) and intense (33.33%; 18/54) estrus response but, the percentage of buffaloes with moderate and intense estrus response was similar (P > 0.05; Table 2).
| Parameter | Percentage of buffaloes | |||
|---|---|---|---|---|
| Group-I (n = 40 ) | Group-II (n = 54) | Overall (n = 94) | ||
| Estrus response | Weak (%) | 52.50bB (21/40) | 12.96aA (7/54) | - |
| Moderate (%) | 40.00b (16/40) | 53.71b (29/54) | - | |
| Intense (%) | 7.50aA (3/40) | 33.33bB (18/54) | - | |
| FSCR (%) vis-à-vis estrus response | Weak (%) | 0.0 (0/21) | 0.0 (0/7) | 0.00 (0/28) |
| Moderate (%) | 50.0 (8/16) | 0.0 (0/29) | 17.78 (8/45) | |
| Intense (%) | 100.0 (3/3) | 100.0 (18/18) | 100.0 (21/21) | |
| FSCR (%) | - | 27.50 (11/40) | 33.33 (18/54) | - |
| a, b (P < 0.05) different superscripts differed significantly from each other within same group; A, B (P < 0.05) different superscripts differed significantly from each other between groups within same intensity of estrus response; n: number of buffaloes (Group-I: d0: PGF2α; Group-II: d0: PGF2α, d1: EB); (EB: Estradiol benzoate; PGF2α: Prostaglandin F2α) | ||||
The percentage of buffaloes with weak estrus response was significantly (P < 0.05) lesser in Group-II (12.96%; 7/54) than Group-I (52.50%; 21/40). Also, the buffaloes showing intense estrus response were significantly (P < 0.05) greater in Group-II (33.33%; 18/54) as compared to Group-I (7.50%; 3/40). However, In case of moderate estrus response the percentage of buffaloes in both groups was similar (P > 0.05; Table 2).
First service conception rate (%)
The FSCR (%) within weak, moderate and intense estrus response were similar (P > 0.05) in both groups. The overall FSCR (%) was also similar (P > 0.05) among different intensity of estrus response (Table 2). The FSCR (%) was higher in Group-II (33.33%) as compared to Group-I (27.5%) but could not reach to the significant level (P > 0.05; Table 2).
First service conception rate (%)
The FSCR (%) within weak, moderate and intense estrus response were similar (P>0.05) in both groups. The overall FSCR (%) was also similar (P>0.05) among different intensity of estrus response (Table 2). The FSCR (%) was higher in Group-II (33.33%) as compared to Group-I (27.5%) but could not reach to the significant level (P>0.05; Table 2).
Ovarian and endocrine profile on first two days of the protocols
The largest follicle (LF) diameter on day 0 and day 1 was significantly greater (P<0.05) in Group-I than Group-II (Table 3). The mean CL area (mm2) on day 0 was found to be significantly (P<0.05) greater in Group-I than Group-II (Table 3). The plasma P4 concentrations on day 0 as well as on day 1 was similar (P>0.05) in both the groups. The plasma E2 (pg/ml) and IGF-1 (ng/ml) concentration on day 0 and day 1 was found to be similar (P>0.05) in both the groups (Table 3).
Ovarian and endocrine profile on day of AI and days 5 and 12 post-AI
On the day of AI, POF diameter (mm) and IGF-1 concentration (ng/ml) was recorded similar (P>0.05) in both the groups (Table 4). However, plasma E2 concentration of Group-II was significantly (P<0.05) higher than Group-I (Table 4). Additionally, on day 5 and 12 post-AI, CL area (mm2) and plasma P4 concentration (ng/ml) were found to be similar (P>0.05) between Group-I and Group-II (Table 4).
Ovarian and endocrine profile on day of AI, and day 5 and 12 post-AI vis-à-vis pregnancy status
On the day of AI, the POF diameter (mm) of Group-I pregnant and non-pregnant buffaloes was similar (P>0.05) than pregnant and non-pregnant buffaloes of Group-II, respectively (Table 5). The plasma E2 concentration (pg/ml) of pregnant and non-pregnant buffaloes of Group-I was significantly (P<0.05) lesser than pregnant and non-pregnant buffaloes of Group-II, respectively. However, plasma E2 concentration of pregnant buffaloes was higher (P<0.05) than non-pregnant buffaloes in Group-II (Table 5). The plasma IGF-1 concentration (ng/ml) was greater (P<0.05) in pregnant than their corresponding non-pregnant buffaloes in Group-I and II (Table 5). The CL area (mm2) of pregnant and non-pregnant buffaloes did not differ significantly (P>0.05) on day 5 and 12 post-AI in Group-I and Group-II (Table 5). The plasma P4 (ng/ml) concentration of pregnant buffaloes on day 5 and 12 post-AI was significantly (P<0.05) greater than non-pregnant counterpart of Group-I and Group-II (Table 5).
Reproductive efficiency is a key factor to ensure profitability in dairy farms. In buffalo farming reduced estrus expression, variable duration of estrus, unpredictable variability in duration of estrous cycle and estrus period remained the major component of declining reproductive performance (Campanile et al. 2010; Mohan et al. 2010; Purohit and Rao 2018) and limits the success of AI programme. To reduce dependency on the detection of visible signs of estrus, the use of timed-AI through ovulation synchronization is being adopted from so long. The results of present study revealed that short duration estrus synchronization protocol with combination of PGF2α and estradiol benzoate resulted in improved FSCR (%) as compared to PGF2α alone. Bandai et al. (2020) used similar short term estrus synchronization protocol with combination of PGF2α and estradiol benzoate in lactating cows during diestrus phase and reported higher FSCR (56.6%) than obtained in present study in estradiol group. This discrepancy in FSCR can be attributed to delayed time of AI used in present study (48h after estradiol benzoate) as compared to 24–28 hours in above mentioned study. As LH concentrations were not measured in both studies but perusal of literature indicated that exogenous estradiol resulted in LH surge approximately 16 to 24h after administration in low progesterone environment and resultant ovulation after 8-36h (average 24-26h) of end of LH surge (Mohan et al. 2009; 2010; Mirmhamoudi et al. 2014) in buffaloes treated with heatsynch and estradoublesynch protocols. Moreover, similar timing for LH surge after estardiol administration during low progesterone environment (14–20 hours) has been reported by Cirit et al. (2008) while using a short term estrus synchronization protocol in lactating cows by using combination of PGF2α, GnRH and estradiol. Moreover, improved FSCR in estradiol group in present study may be due to improved estrus response, higher estradiol concentration and marginally greater CL area leading to better hormonal profile post ovulation.
In present study, number of buffaloes exhibited weak estrus response are higher in Group-I as compared to Group-II while, number of buffalos with intense estrus response were significantly higher in Group-II and this fact can be attributed to higher pregnancy rate observed in Group-II. Numerous studies have reported improved fertility outcomes with increased intensity of estrus expression are related to improved pregnancy outcome (Rodrigues et al. 2018; Madureira et al. 2015). Moreover, retrospective analysis of data revealed that during present study, significantly higher FSCR was obtained in buffaloes exhibited intense estrus response irrespective of treatment group and it further strength the fact that better estrus expression leads to more pregnancy rate. It may be speculated that improved estrus response resulted in better coordination of events culminating in timely ovulation leading to improved fertility. Lowered progesterone levels with elevated estradiol levels on day of AI were determined as probable indicators of fertility in animals undergoing timed-AI protocols (Bello et al. 2006; Saini et al. 2023). It has been postulated that lowered progesterone in conjugation with elevated estradiol resulted in timely LH surge which is essential to cause timely ovulation and effects competence of oocyte (Greenwald 1994). Consistent to above observation, in present study lowered progesterone coupled with elevated estradiol was observed in Group-II animals which further strengthen our speculation that higher intensity of estrus response resulted in improved endocrine profile at ovulation leading to improved luteal function post ovulation and better fertility outcomes.
POF size at time of AI was found to effect hormonal profile before and after ovulation and in-turn effecting pregnancy outcomes post-AI in buffalo (Pandey et al. 2011; 2018) and cattle (Bello et al. 2006; Pursley and Martins 2011). The increasing size of POF was associated with increased estradiol concentrations on day of AI, improved luteal function and higher fertility rates post-AI. But, the retrospective analysis of data in present study revealed that pregnant buffaloes of both groups have ovulated similar sized POF as compared to non-pregnant animals but, the circulatory concentration of Estradiol was found greater in pregnant animals of Group-II only than their corresponding non-pregnant. Moreover, circulatory concentrations of IGF-1 were also found to be higher in pregnant animals as compared to non-pregnant animals indicating that increased circulatory concentration of estradiol and IGF-1 together might have played role in leading to culmination of ovulatory mechanism that is prerequisite for improved luteal profile post-ovulation. Increased levels of IGF-1 was associated with improved quality of follicles, improved oocyte competence by amplifying action of gonadotrophins on follicles (Behl and Kaul 2002). Additionally, IGF-1 was also found to stimulate adequate maturation of oocyte and is also required for timely ovulation (Yoshimura 1998). Thus, it may be speculated that, during present study, elevated levels of IGF-1 observed in pregnant buffaloes led to improved oocyte competence and resulted in timely ovulation after adequate maturation. Moreover, a positive constructive relation observed between estradiol and IGF-1 concentrations on day of AI further strengthen the fact that improved hormonal milieu at time of AI is an important aspect for increased conception rates.
The previous studies including dairy cows (Pursely and Martins 2011) and buffalo (Pandey et al. 2011; 2018) had constructive relationship between POF diameter and luteal profiles. Many studies observed that P4 concentartion post-AI affected the pregnancy status as increased plasma P4 concentration post-AI was conducive for establishment of pregnancy in cows (Grimard et al. 2006; Pursely and Martins 2011) and buffalo (Pandey et al. 2011; 2018). On the lines of these observation, in present study higher progesterone concentration on day 5 and 12 post-AI was found in pregnant animals of both groups. It has been observed that sufficient levels of progesterone concentrations are necessary for normal uterine function, supports growth of conceptus and prevents embryo loss during phases of rapid embryonic growth between day 8 to 16 post-AI (Diskin and Morris 2007).
Based on the finding of the study it was concluded that short term estrus synchronization protocol with administration of Estradiol benzoate 24h after PGF2α improved the estrus response, plasma estrogen concentration at AI with better post-ovulation luteal profile in cyclic Murrah buffaloes. This protocol yielded almost similar pregnancy rates as resulted in longer traditional protocols but, ease of less injections administration and cheaper cost could make it more practical. More studies consisting of large number of animals are warranted.
Acknowledgements The authors are grateful to the management team of the animal farm for their unconditional help through the whole course of investigation.
Author contribution Vinay Yadav: conceptualization, investigation, writing, analysis, and drafting of the manuscript. Anand Kumar Pandey: conceptualization, resources, analysis, review, and editing of the manuscript. Sandeep Kumar: conceptualization, analysis, resources, review, and editing of the manuscript. Gitesh Saini: investigation, analysis, and drafting of the manuscript. Dipin Chander Yadav: conceptualization and methodology. Amarjeet Bisla: statistical analysis.
Data availability The datasets generated during and/or analysed during the current study will be made available by the corresponding author on reasonable request.
Funding The authors declare that no funds, grants, or other support were received for this study.
Ethics approval Present investigation was conducted after prior approval from Institutional Animal Ethics Committee (Registration: 1669/GO/ReBiBt-S/Re-L/12/CPCSEA), COVS, LUVAS (Hisar) vide letter number- VCC/IAEC/2022/624-51; dated- 10-05-2022.
Consent to participate The authors have permission to participate.
Consent for publication The authors have permission for publication.
Competing interests The authors declare no conflict of interest.
- Atabay, E.C., Maylem, E.R.S., Salazar, R.L., 2020. Enhancing prostaglandin-based estrus synchronization protocol for artificial insemination in water buffaloes. Buffalo Bulletin, 39(1), 53-60.
- Bandai, K., Kusaka, H., Miura, H., Kikuchi, M., Sakaguchi, M., 2020. A simple and practical short-term timed artificial insemination protocol using estradiol benzoate with prostaglandin F2α in lactating dairy cows. Theriogenology, 141, 197-201.
- Baruselli, P.S., 2001. Control of follicular development applied to reproduction biotechnologies in buffalo. In: Proceedings of the I Congresso Nazionale sull’allevamento del Bufalo. Book of the Congress, p. 128–46.
- Behl, R., Kaul, R., 2002. Insulin like growth factor 1 and regulation of ovarian function in mammals. Indian Journal of Experimental Biology, 40, 25-30.
- Bello, N.M., Steibel, J.P., Pursley, J.R., 2006. Optimizing ovulation to first GnRH improved outcomes to each hormonal injection of Ovsynch in lactating dairy cows. Journal of Dairy Science, 89(9), 3413-3424.
- Campanile, G., Baruselli, P.S., Neglia, G., Vecchio, D., Gasparrini, B., Gimenes, L.U., Michael, J.D., 2010. Ovarian function in the buffalo and implications for embryo development and assisted reproduction. Animal Reproduction Science, 121(1-2), 1-11.
- Cirit, Ü., Bacinoglu, S., Tas¸ M., Demir, K., Bas¸ A., Ak, K., 2008. Evaluation of short estrus synchronization methods in dairy cows. Animal Reproduction Science, 109, 65-76.
- Dhaka, A.P., Phogat, J.B., Singh, S., Pandey, A.K., Sharma, K., Kumari, S., 2019. Efficacy of modified co-synch plus protocol with or without progesterone device for estrus induction and conception rate in Murrah buffaloes under field conditions during summer season. Buffalo Bulletin, 38(2), 353-361.
- Diskin, M.G., Morris, D.G., 2008. Embryonic and early foetal losses in cattle and other ruminants. Reproduction in Domestic Animals, 43, 260–267.
- Greenwald, G.S., 1994. Follicular development and its control. The physiology of reproduction, 1, 629-724.
- Grimard, B., Freret, S., Chevallier, A., Pinto, A., Ponsart, C., Humblot, P., 2006. Genetic and environmental factors influencing first service conception rate and late embryonic/foetal mortality in low fertility dairy herds. Animal Reproduction Science, 91(1-2), 31-44.
- Jeong, J.K., Kang, H.G., Hur, T.Y., Kim, I.H., 2013. Synchronization using PGF2α and estradiol with or without GnRH for timed artificial insemination in dairy cows. Journal of Reproduction and Development, 59(1), 97-101.
- Layek, S.S., Mohanty, T.K., Kumaresan, A., Behera, K., Chand, S., 2011. Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle. Animal Reproduction Science, 129(3-4), 140-145.
- Madan, M., Prakash, B.S., 2007. Reproductive endocrinology and biotechnology applications among buffaloes. Society of Reproduction and Fertility, 64, 261–81.
- Madureira, A.M.L., Silper, B.F., Burnett, T.A., Polsky, L., Cruppe, L.H., Veira, D.M., Cerri, R.L.A., 2015. Factors affecting expression of estrus measured by activity monitors and conception risk of lactating dairy cows. Journal of Dairy Science, 98(10), 7003-7014.
- McArt, J.A.A., Caixeta, L.S., Machado, V.S., Guard, C.L., Galvao, K.N., Sa Filho, O.G., 2010. Ovsynch versus Ultrasynch: reproductive efficacy of a dairy cattle synchronization protocol incorporating corpus luteum function. Journal of Dairy Sciences, 93, 2525-32.
- Mirmahmoudi, R., Prakash, B.S., 2012. The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis). Gen. Comp. Endocrinol, 177(1), 153-159.
- Mirmahmoudi, R., Souri, M., Prakash, B.S., 2014. Endocrine changes, timing of ovulation, ovarian follicular growth and efficacy of a novel protocol (Estradoublesynch) for synchronization of ovulation and timed artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology, 81(2), 237-242.
- Mohan, K., Kumar, V., Sarkar, M., Prakash, B.S., 2010. Temporal changes in endogenous estrogens and expression of behaviors associated with estrus during the periovulaory period in Murrah buffaloes (Bubalus bubalis). Tropical Animal Health and Production, 42(1), 21-26.
- Mohan, K., Sarkar, M., Prakash, B.S., 2009. Efficiency of heatsynch protocol in estrous synchronization, ovulation and conception of dairy buffaloes (Bubalus bubalis). Asian-australas. Journal of Animal Science, 22(6), 774-780.
- Pandey, A.K., Dhaliwal, G.S., Ghuman, S.P.S., Agarwal, S.K., 2011. Impact of preovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis). Animal Reproduction Science, 123(3-4), 169–174.
- Pandey, A.K., Ghuman, S.P.S., Dhaliwal, G.S., Honparkhe, M., Phogat, J.B., Kumar, S., 2018. Effects of preovulatory follicle size on estradiol concentrations, corpus luteum diameter, progesterone concentrations and subsequent pregnancy rate in buffalo cows (Bubalus bubalis). Theriogenology, 107, 57–62.
- Paul, V., Prakash, B.S., 2005. Efficacy of the ovsynch protocol for synchronization of ovulation and fixed-time artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology, 64, 1049–60.
- Purohit, G.N., Rao, T.K., 2018. Estrus detection in buffaloes. Bubaline Theriogenology, Ithaca, NY: International Veterinary Information Service, 1-26.
- Pursley, J.R., Martins, J.P.N., 2011. Impact of circulating concentrationsconc. Of progesterone and antral age of the ovulatory follicle on fertility of high-producing lactating dairy cows. Reproduction Fertility Development, 24(1), 267-271.
- Rodrigues, A.D., Cooke, R.F., Cipriano, R.S., Silva, L.G.T., Cerri, R.L.A., Cruppe, L.H., Vasconcelos, J.L.M., 2018. Impacts of estrus expression and intensity during a timed-AI protocol on variables associated with fertility and pregnancy success in Bos indicus-influenced beef cows. Journal of Animal Science, 96(1), 236-249.
- Saini, G., Kumar, S., Pandey, A.K., Singh, H., Virmani, M., (2023). Presynchronization with simultaneous administration of GnRH and prostaglandin F2α (PGF2α) 7 days prior to Ovsynch improves reproductive profile in Hariana zebu cow. Tropical Animal Health and Production, 55(1), 19.
- Sharma, K., Phogat, J.B., Pandey, A.K., Dhaka, A.P., Singh, S., Ghadwal, S., 2021. Role of progesterone supplementation in estrus induction in Murrah buffaloes under field conditions in non-breeding season. Buffalo Bulletin, 40(1), 99-106.
- Singh, H., Pandey, A.K., Kumar, S., Saini, G., Duggal, R., Bangar, Y.C., Kumar, H., 2022. 5d CIDR-Heatsynch improves the circulatory estradiol levels, estrus expression and conception rate in anestrus buffalo (Bubalus bubalis). Animal Biotechnology, 1-12.
- Yoshimura, Y., 1998. Insulin‐like growth factors and ovarian physiology. Journal of Obstetrics and Gynaecology Research, 24(5), 305-323.