Research Article

Incorporation of CIDR into Ovsynch Protocol Improved the Reproductive Performance of Anestrous Buffaloes during Low Breeding Season

Gokarna Gautam1*, Hari Adhikari1, Bhuminand Devkota1, Subir Singh2

1Department of Theriogenology, Faculty of Animal Science Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Bharatpur-15, Chitwan, Nepal; 2Department of Veterinary Medicine and Public Health, Faculty of Animal Science Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Bharatpur-15, Chitwan, Nepal.

Received | January 22, 2024; Accepted | March 05, 2024; Published | April 08, 2024

*Correspondence | Gokarna Gautam, Department of Theriogenology, Faculty of Animal Science Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Chitwan, Nepal; Email: ggautam@afu.edu.np

Citation | Gautam G, Adhikari H, Devkota B, Singh S (2024). Incorporation of CIDR into ovsynch protocol improved the reproductive performance of anestrous buffaloes during low breeding season. Adv. Anim. Vet. Sci., 12(6):994-1001.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.6.994.1001

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Buffalo (Bubalus bubalis) can serve as a reliable living bank in Asian countries as the poor farmer can have their buffalo as an easily convertible currency to serve their immediate needs with optimum benefit through milk, meat, hide, manure, draught power and even the horn and hair (Nanda et al., 2003). The buffalo is the most important livestock species in Nepalese agricultural system as it contributes 57.2% of the total milk production and 36.1% of the total meat production in the nation (MoALD, 2022). However, the milk production efficiency of buffaloes is quite low mainly due to sub-fertility and infertility characterized by delayed sexual maturity, prolonged postpartum anestrous period, poor estrus expression and seasonal breeding pattern (Shah et al., 1990; Nanda et al., 2003; Barile, 2005; Devkota and Bohora, 2009; Kumar et al., 2012; Chaudhari et al., 2012).

Seasonality of reproduction was the characteristic feature of the buffaloes in the countries away from the equator, which was characterized by the active breeding activities during the months of short day length (fall and winter) and the cessation of sexual activity during the months of longer day length (summer) also known as summer anestrus (Qureshi et al., 1999; Neglia et al., 2003; Presicce et al., 2004; De Rensis and Lopez-Gatius, 2007; Zicarelli, 2007; Devkota and Bohora, 2009; Das and Khan, 2010; Kumar et al., 2012; D’Occhio et al., 2020). Seasonal breeding pattern was the one of the major causes of poor reproductive efficiency in Nepalese buffaloes with April to June as the low breeding season (Devkota and Bohora, 2009).

A number of hormonal protocols used in dairy cows have been tried to improve reproductive performance of anestrous buffaloes during low breeding season, however with poor to moderate efficacy (De Rensis et al., 2005; Sah and Nakao, 2010; Carvalho et al., 2011, 2013; Devkota et al., 2013; Warriach et al., 2015). As in cattle, the Ovsynch protocol is one of the most commonly used ovulation synchronization protocols in buffalo that allows fixed time artificial insemination (Paul and Prakash, 2005; Carvalho et al., 2007, 2013; Warriach et al., 2008, 2015) however, the effectiveness of it depends on the status of ovarian cyclicity (Pursley et al., 1997; Moreira et al., 2000; Stevenson et al., 2006; Souza et al., 2007). Ovsynch protocol was effective specially in cyclic animals (Devkota and Bohora, 2009; Nowicki et al., 2017) but less effective in non-cyclic animals (De Rensis et al., 2005; Karen and Darwish, 2010; Malik et al., 2011). For the treatment of anestrous buffaloes with inactive ovaries, the progesterone-releasing intravaginal devices such as CIDR (controlled internal drug release) and PRID (progesterone releasing intravaginal device) have been used in conjunction with Ovsynch regimens (Carvalho et al., 2011; Barile, 2012; Yendraliza et al., 2015). Although Ovsynch was tried in Nepalese buffaloes during good breeding season (Kharel et al., 2017; Shah et al., 2017; Devkota et al., 2021) and CIDR-synch (incorporation of CIDR in Ovsynch) tried during low breeding season (Devkota et al., 2021; Lamsal et al., 2017), the studies comparing the efficacy of Ovsynch vs CIDR-synch in anestrous buffaloes during low breeding season in Nepalese water buffaloes were lacking. As most of the buffaloes during low breeding season are non-cyclic (Singh et al., 2000; Nanda and Nakao, 2003), in the present study it was hypothesized that the incorporation of CIDR in the Ovsynch protocol would be effective to resume estrous cyclicity and to improve reproductive performance in anestrous buffaloes during low breeding season. Therefore, the objectives of the present study was to determine the effectiveness of CIDR-synch protocol as compared to the standard Ovsynch protocol for the treatment of anestrus in buffaloes during low breeding season.

MATERIALS AND METHODS

The use of animals in this study was in accordance with the ARRIVE (Animal research: Reporting of in vivo Experiments) guidelines.

Animals

This study was conducted in anestrous Murrah cross-bred buffaloes from six commercial buffalo farms located in Chitwan district, which is a plain region in the mid southern part of Nepal and it has a sub-tropical climatic condition with hot humid summer and cool dry winter. Only the buffaloes that were beyond 70 days postpartum with no anatomical defects or anomalies in their reproductive tract, that did not express estrus since last calving and that had body condition score (BCS) >2.25 were included in the trial. All the buffaloes were reared in similar management conditions with 24 hours tie-stall barn, hand milking twice daily and feeding seasonally available roughages and farm-prepared concentrate feed consisting of maize, rice bran, wheat bran, mustard oil-cake and commercial vitamin-mineral mixtures. The purposive sampling technique was used for selecting buffaloes until the number of buffaloes reached to 60. Parity of buffaloes (mean ± S.E.) was 3.35 ± 1.31 (Range: 1-8). Systemic recording of milk yield was not kept by the owners. Based on the approximate values provided by the buffalo owners, the milk yield of the buffaloes in the experiment ranged from 4 to 9 liters per day.

Buffaloes in the trial were clinically examined to assess their BCS (1-5 scale with 0.25 increment, Ferguson et al., 1994). BCS (mean ± S.E.) was 3 ± 0.45 (Range: 2.5-4). Transrectal ultrasonography (Ebit 30VET, CHISON Medical Technologies Co., Ltd, China) of reproductive organs was performed using 10 MHz transducer to determine the ovarian cyclicity status and to rule out the anatomical defects or anomalies of the reproductive system, if any. All the buffaloes in the study were drenched with broad-spectrum anthelmintic (oxyclozanide + levamisole, Zanide L Forte, QMED Pharmaceuticals, Nepal) at the recommended doses. After deworming, the mineral-vitamin mixture supplement (Minfa Gold- Intas Pharmaceuticals, India) was provided to all the buffaloes at the dose rate of 50 gm per animal per day for 20 days. After that the hormonal protocols were applied.

Hormonal treatment protocols

Five buffaloes were excluded before the commencement of hormonal protocols because of owners unwillingness to include those buffaloes in the trial. Remaining anestrous buffaloes (n= 55) were randomly divided into two treatment groups (Figures 1, 2). In Group T1 (Ovsynch, n= 25), Ovsynch protocol was applied in which the buffaloes were injected intramuscular (i.m.) with GnRH analogue, buserelin acetate 20 µg (Gynarich, Intas Pharmaceuticals, India) on a random day considered as day zero (d0), PGF2α analogue, cloprostenol 500 µg (Cloprochem, Interchem, Netherlands) on d7, followed by second GnRH injection on d9 and fixed timed artificial insemination (FTAI) done at 16-20 hours after the second GnRH injection. In Group T2 (CIDR-synch, n=30), all the buffaloes were treated as in T1 except a CIDR (Controlled Internal Drug Release, Eazi-Breed, Zoites, Australia) device containing 1.9 gm of progesterone was inserted into the vagina of each buffalo on d0 and removed on d7 at the time of PGF2α injection. All of the buffaloes in both treatment groups were inseminated artificially by the first author using frozen thawed semen of Murrah buffaloes from the National Livestock Breeding Office, Pokhara, Nepal.

 

 

Estrus detection

Starting after PGF2α injection until FTAI, the buffaloes in both treatment groups were observed for the external estrous signs (mucus discharge from vulva, bellowing, vulva swelling, teat engorgement and decrease in milk yield) at least three times a day. Estrus related changes in the reproductive organs (uterine contraction) were determined at the time of FTAI. Buffalo was considered to be in estrus if it showed any of the external estrous signs or uterine contraction.

Ovulation confirmation and corpus luteum (CL) size determination

Ovulation was confirmed based on the presence of CL on 8-9 days after FTAI examined by transrectal ultrasonography (Ebit 30VET, CHISON Medical Technologies Co., Ltd, China) using 10 MHz transducer. Two diameters of CL at perpendicular to each other were measured and the average of two measurements was taken as the diameter of CL.

Pregnancy diagnosis

Early pregnancy diagnosis was conducted during 30-35 days after FTAI using transrectal Ultrasonography. Final pregnancy was confirmed during 75-90 days after FTAI using transrectal palpation. Pregnancy loss between first and second examination if any, was recorded.

Statistical analyses

The data were analyzed using IBM SPSS statistical software (version 26). Estrus expression rate, ovulation rate and pregnancy rates between two treatment groups were compared using Chi-square test; when the expected frequency was less than 5 in more than 20% of the cells, the Fisher’s exact probability test was used. Effect of ovarian status at the initiation of the protocol on pregnancy outcome from FTAI was also analyzed using Chi-square test or Fisher’s exact probability test, whichever applicable. The results with P-value ≤0.05 were considered as significant whereas those with 0.05<P≤ 0.1 were considered to have tendency effect.

RESULTS

Estrus expression and ovulation rates

Table 1 shows the proportions of buffaloes showing various estrous signs. Among various estrous signs, the proportions of buffaloes showing mucus discharge (P=0.01), those having uterine contraction during FTAI (P=0.02) and those with decrease in milk yield (P=0.1) were higher in CIDR-synch than in Ovsynch groups. Ultimately, the overall estrus expression rate was higher (P=0.008) in CIDR-synch group (96.7%) than in Ovsynch group (68%).

 

Table 1: Proportions of buffaloes showing various estrous signs in Ovsynch and CIDR-synch groups.

Estrous signs	Total (n=55)	Treatment group		P value
		Ovsynch (n=25)	CIDR-synch (n=30)
Mucus discharge (%)	54.5	36.0	70.0	0.01
Bellowing (%)	14.5	16.0	13.3	1
Vulva swelling (%)	32.7	28.0	36.7	0.50
Teat engorgement (%)	43.6	44.0	43.3	1
Decrease in milk yield (%)	63.6	52.0	73.3	0.10
Uterine contraction during FTAI (%)	78.2	64.0	90.0	0.02
Overall estrous expression rate (%)	83.6	68.0	96.7	0.008

 

Table 2: Pregnancy rate of FTAI and the overall pregnancy rate (resulting from FTAI plus natural breeding of buffaloes returning to estrus within one month of FTAI) in Ovsynch and CIDR-synch groups.

Parameters	Overall	Treatment groups		P value
		Ovsynch	CIDR-synch
No. of buffaloes treated	55	25	30
No. of buffaloes pregnant from FTAI (A)	14	4	10
FTAI pregnancy rate (%)	25.5	16.0	33.3	0.14
No. of buffaloes returned to estrus within one month of FTAI and bred with bull	14	3	11
No. of buffaloes pregnant from natural breeding within one month of FTAI (B)	5	1	4
Overall pregnancy rate within one month of FTAI (A+B) (%)	34.5 (19/55)	20.0 (5/25)	46.7 (14/30)	0.04

 

Table 3: Effect of ovarian status at the initiation of the protocol on pregnancy outcome from FTAI in Ovsynch and CIDR-synch protocol treated buffaloes.

Protocol	Ovarian status at the initiation of the protocol	No. of buffaloes (n=55)	Pregnancy rate (%)	P value
Ovsynch	Cyclic (CL present)	9	1/9 (11.1)	1
	Non-cyclic (CL absent)	16	3/16 (18.8)
CIDR-synch	Cyclic (CL present)	10	3/10 (30.0)	1
	Non-cyclic (CL absent)	20	7/20 (35.0)

 

Overall, 85.5% (47/55) buffaloes had ovulation (presence of CL on 8-9 day after FTAI). There was a tendency (P=0.1) that the ovulation rate (93.3% vs 76.0%) was higher in CIDR-synch group (28/30) than in Ovsynch group (19/25).

Pregnancy rates

There was no pregnancy loss during a period between 30-35 days and 75-90 days post FTAI. Table 2 shows the pregnancy outcomes and the resumption of estrous cyclicity within 40 days of treatment in Ovsynch and CIDR-synch treated buffaloes. Pregnancy rates from FTAI did not differ (P= 0.14) between Ovsynch (16%) and CIDR-synch (33.3%) groups. However, the overall pregnancy rate within one month after FTAI (resulting from FTAI + natural breeding within one month of FTAI) was significantly (P= 0.04) higher in CIDR-synch (46.7%) than in Ovsynch (20%) groups.

Effect of ovarian status at the initiation of the protocol on pregnancy outcome

Effect of ovarian status at the initiation of the protocol on pregnancy outcome from FTAI has been shown in Table 3. There was no significant effect of ovarian status at the initiation of the protocol on pregnancy outcome from FTAI both in Ovysnch and CIDR-synch groups.

discussion

This study was conducted to determine the effectiveness of incorporation of CIDR in Ovsynch protocol for the treatment of anestrous buffaloes during low breeding season (April to June). One group of anestrous buffaloes received standard Ovsynch protocol while in another group (CIDR-synch) of anestrous buffaloes, in addition to Ovsynch protocol, a CIDR was inserted for seven days (from d0 to d7). There was significant difference between two treatment groups in terms of estrus expression. Since all the buffaloes in this experiment were in 24 hrs tie-stall system, the estrus expression after treatment was based on the observation of secondary estrous signs (mucus discharge from vulva, bellowing, vulva swelling, teat engorgement, decrease in milk yield and uterine contraction during FTAI). In total, the highest proportion of buffaloes showed the uterine contraction during FTAI (78.2%) followed by the decrease in milk yield (63.6%), mucus discharge (54.5%), teat engorgement (43.6%), swelling of vulva (32.7%) and bellowing (14.5%). The proportions of buffaloes showing mucus discharge, uterine contraction during FTAI, decrease in milk yield and ultimately the overall estrus expression rate were higher in CIDR-synch group than in Ovsynch group. The proportions of buffaloes showing estrus in CIDR-synch group in the present study (96.7%) was almost similar to the findings of previous studies that used CIDR-synch protocol in buffaloes: 92% (Naseer et al., 2011) and 100% (Kajaysri et al., 2015). Likewise, the estrous expression in the Ovsynch group in the present study (68%) was also similar to the findings of a previous study (73.9%) in which the buffaloes were subjected to Ovsynch protocol during low breeding season (Abulaiti et al., 2022). Although expression of estrous signs is generally not mandatory in FTAI protocols, previous studies in cattle have shown that the pregnancy outcome in FTAI was better in cows displaying estrus than in cows without estrous signs (Loeffler et al., 1999; Pereira et al., 2016).

Ovulation was determined based on the presence of corpus luteum during 8-9 days after FTAI. Overall ovulation rate was 85.5% though it was not known when the ovulation actually took place; determination of exact time of ovulation was beyond the scope of this study. There was a tendency that the ovulation rate was higher in buffaloes treated with CIDR-synch than in buffaloes treated with Ovsynch protocols. Ovulation rate in CIDR-synch group in the present study (93.3%) was almost similar to that (89%) reported by a previous study in which buffaloes were treated with CIDR (7 days)-PG-GnRH protocol (Naseer et al., 2011). Likewise, the ovulation rate in Ovsynch group in the present study (76%) was almost similar to the findings (60.9%) of Abulaiti et al. (2022). Rapid drop in circulatory progesterone concentration following CIDR removal might be the cause for higher estrous expression rate and ovulation rate in CIDR-synch group as compared to Ovsynch group. This rapid drop in progesterone concentration removes the negative feedback effect of progesterone on hypothalamus, as a result there is release of GnRH from hypothalamus followed by the release of FSH and LH from anterior pituitary, and the subsequent restoration of ovarian cyclicity (Zerbe et al., 1999; Zabeel et al., 2009; Azawi et al., 2012).

Pregnancy rate of FTAI in CIDR-synch group (33.3%) in the present study was almost similar to the findings of previous studies that used CIDR-synch [31.8% (Azawi et al., 2012), 32.8% (El-Tarabany, 2016)] and CIDR co-synch protocols (37% (Naseer et al., 2011)). Pregnancy outcomes of FTAI in Ovsynch protocol during low breeding season in buffaloes were inconsistent in previous studies that ranged from 0% (Jabeen et al., 2013) to 26.1% (Abulaiti et al., 2022) and 29.4% (Warriach et al., 2008). Although the pregnancy rate from FTAI in the present study did not differ between Ovsynch and CIDR-synch groups, the overall pregnancy rate (resulting from FTAI + natural breeding within one month of FTAI) was significantly higher in CIDR-synch group (46.7%) than in Ovsynch group (20%). This increase in overall pregnancy rate within one month of FTAI in CIDR-synch treated buffaloes might be attributed to the higher proportion of buffaloes that resumed estrous cyclicity due to hormonal protocol. It has been shown that the progesterone concentration in previous luteal phase can affect follicle development and subsequent fertility along with the sufficient priming of the endometrium (Stevenson et al., 2006; Madureira et al., 2021). It has been also demonstrated that progesterone increases the expression of estradiol receptors in the hypothalamus (Van Eerdenburg et al., 2000; Gumen and Wiltbank, 2002) and thus, primes the hypothalamus to be more responsive to estradiol (Woelders et al., 2014) leading to the resumption of estrous cyclicity. Thus, the present study clearly indicated that the intravaginal progesterone based treatments were effective to resume estrous cyclicity and to enhance the subsequent reproductive performance in anestrous buffaloes during low breeding season.

Although previous study demonstrated that the pregnancy outcome from estrus synchronization protocol in cattle was affected by the presence of CL or dominant follicle at the initiation of the protocol (Stevenson et al., 2012), in the present study, there was no effect of ovarian cyclicity status at the initiation of the protocol on pregnancy outcome from FTAI in both treatment groups of buffaloes. This might be due to small number of buffaloes used in this study. Thus, further detailed study might be needed to rule out the effect of ovarian cyclicity status, if any, on the efficacy of estrous synchronization protocol in anestrous buffaloes during low breeding season.

CONCLUSIONs

Incorporation of CIDR into Ovsynch protocol improved the resumption of estrous cyclicity, ovulation and overall pregnancy outcome within one month in anestrous buffaloes during low breeding season. During the revision of our manuscript (addressing the reviewer’s comments), we forgot to delete this sentence, although we removed it from ABSTRACT. As the results related to this statement have been deleted, it is not logical to keep this statement in conclusion.

ACKNOWLEDGMENT

This study was funded by the University Grant Commission (UGC) Nepal; the authors are thankful to the UGC Nepal for its financial support (Collaborative Research Grant (CRG-75/76-Ag and F-1)). The authors would like to acknowledge the buffalo owners for allowing to use their buffaloes for this study.

NOVELTY STATEMENT

This study demonstrated that the inclusion of CIDR in Ovsynch protocol improved the reproductive performance of anestrous buffaloes during low breeding season through the resumption of estrous cyclicity and ovulation.

AUTHOR’S CONTRIBUTION

GG: Conceptualization, funding acquisition, project administration, resources, investigation, methodology, formal analysis, supervision, writing-original draft, writing review and editing. HA: Methodology, data curation, formal analysis, writing original draft. BD: Methodology, investigation, supervision, writing review and editing. SS: Methodology, investigation, supervision.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Abulaiti A, Naseer Z, Ahmed Z, Wang D, Hua G, Yang L (2022). Effect of different synchronization regimens on reproductive variables of crossbred (swamp× riverine) nulliparous and multiparous buffaloes during peak and low breeding seasons. Animals, 12(4): 415. https://doi.org/10.3390/ani12040415

Azawi OI, Ali MD, Ahmed OS, Al-Hadad AS, Abeh-Jamil MS, Hussien ASA (2012). Treatment of anestrous in Iraqi buffaloes using ovsynch alone or in combination with CIDR. J. Adv. Vet. Res., 2(2): 68-72.

Barile VL (2005). Improving reproductive efficiency in female buffaloes. Livest. Prod. Sci., 92: 183–194. https://doi.org/10.1016/j.livprodsci.2004.06.014

Barile VL (2012). Technologies related with the artificial insemination in buffalo. J. Buff. Sci., 1(2): 139-146. https://doi.org/10.6000/1927-520X.2012.01.02.02

Carvalho N, Soares J, Reis E, Baruselli P (2011). Use of DIB and CRESTAR to synchronize ovulation and FTAI in buffalo heifers during the non-breeding season. Acta Sci. Vet., 16: 390-394.

Carvalho NA, Soares JG, Porto Filho RM, Gimenes LU, Souza DC, Nichi M, Sales JS, Baruselli PS (2013). Equine chorionic gonadotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the nonbreeding season. Theriogenology, 79(3): 423-428. https://doi.org/10.1016/j.theriogenology.2012.10.013

Carvalho NA, Vannucci FS, Amaral R, Baruselli PS (2007). Use of GnRH to induce an accessory corpus luteum in buffaloes fixed time artificially inseminated. Ital. J. Anim. Sci., 6(sup2): 655-658.

Chaudhari BK, Singh JK, Singh M, Maurya PK, Singh AK (2012). Management of reproductive performance in buffalo during summer season. Wayamba J. Anim. Sci., 4: 499-512.

Chaudhari R, Mehrotra S, Krishnaswamy N, Ajevar G, Chaudhary J, Kumar A (2015). Effect of season on the incidence of estrus and calving in cattle and buffaloes. Indian Vet. J., 92: 24–26.

D’Occhio MJ, Ghuman SS, Neglia G, Valle GD, Baruselli PS, Zicarelli L, Visintin JA, Sarkar M, Campanile G (2020). Exogenous and endogenous factors in seasonality of reproduction in buffalo: A review. Theriogenology, 150: 186-192. https://doi.org/10.1016/j.theriogenology.2020.01.044

Das GK, Khan FA (2010). Summer anoestrus in buffalo. A review. Reprod. Domest. Anim., 45(6): e483-e494. https://doi.org/10.1111/j.1439-0531.2010.01598.x

De Rensis F, López-Gatius F (2007). Protocols for synchronizing estrus and ovulation in buffalo (Bubalus bubalis): A review. Theriogenology, 67: 209–216. https://doi.org/10.1016/j.theriogenology.2006.09.039

DeRensis F, Ronci G, Guarneri P, Nguyen BX, Presicce GA, Huszenicza G, Scaramuzzi RJ (2005). Conception rate after fixed time insemination following ovsynch protocol with and without progesterone supplementation in cyclic and non-cyclic mediterranean Italian buffaloes (Bubalus bubalis). Theriogenology, 63: 1824–1831. https://doi.org/10.1016/j.theriogenology.2004.07.024

Devkota B, Bohara TP (2009). Effects of season on pregnancy rates in water buffaloes of Southern Nepal evaluated by using different estrus synchronization protocols during active season and low breeding season. Pak. J. Zool. Suppl., 9: 763-770.

Devkota B, Bohara TP, Yamagishi N (2012). Seasonal variation of anestrus conditions in Buffaloes (Bubalus bubalis) in Southern Nepal. Asian J. Anim. Vet. Adv., 7(9): 910-914. https://doi.org/10.3923/ajava.2012.910.914

Devkota B, Nakao T, Kobayashi K, Sato H, Sah SK, Singh DK, Dhakal IP, Yamagishi N (2013). Effects of treatment for anestrus in water buffaloes with PGF2 alpha and GnRH in comparison with vitamin-mineral supplement, and some factors influencing treatment effects. J. Vet. Med. Sci., 75: 1623-1627. https://doi.org/10.1292/jvms.12-0515

Devkota B, Shah S, Lamsal D, Gautam G (2021). Understanding infertility issues and its management techniques in Nepalese buffalo. In: Book of abstracts of 10th Asian buffalo congress; Agriculture and Forestry University and Ministry of Agriculture and Livestock Development, Nepal: Chitwan, Nepal, October 25-29, pp. 30.

El-Tarabany MS (2016). The efficiency of new CIDR and once-used CIDR to synchronize ovulation in primiparous and multiparous Holstein cows. Anim. Reprod. Sci., 173: 29–34. https://doi.org/10.1016/j.anireprosci.2016.08.006

Ferguson JD, Galligan DT, Thomsen N (1994). Principal descriptors of body condition score in Holstein cows. J. Dairy Sci., 77: 2695-2703. https://doi.org/10.3168/jds.S0022-0302(94)77212-X

Gümen A, Wiltbank MC (2002). An alteration in the hypothalamic action of estradiol due to lack of progesterone exposure can cause follicular cysts in cattle. Biol. Reprod., 66(6): 1689-1695. https://doi.org/10.1095/biolreprod66.6.1689

Jabeen S, Anwar M, Andrabi SMH, Mehmood A, Murtaza S, Shahab M (2013). Determination of Ovsynch efficiency for estrus synchronization by plasma LH and P4 levels in Nili Ravi buffalo during peak and low breeding seasons. Pak. Vet. J., 33: 221-224.

Kajaysri J, Chumchoung C, Photikanit G (2015). Estrus and ovulation responses in anestrus postpartum swamp buffaloes following synchronization with a controlled internal drug release device and prostaglandin F2α based protocols. Buff. Bull., 34(3): 357-368.

Karen AM, Darwish SA (2010). Efficacy of Ovsynch protocol in cyclic and acyclic Egyptian buffaloes in summer. Anim. Reprod. Sci., 119(1-2): 17-23. https://doi.org/10.1016/j.anireprosci.2009.12.005

Kharel CN, Devkota B, Sah SK, Karki RK (2017). Evaluation of ovsynch protocol on reproductive performance of anestrous buffaloes during breeding season in Chitwan, Nepal. Nepalese Vet. J., 34: 41-50. https://doi.org/10.3126/nvj.v34i0.22902

Kumar S, Malik RK, Sharma RK, Dutt R, Singh P, Singh G, Virmani M (2012). Effect of Ovsynch protocol in different hormonal combinations on follicular dynamics in anoestrus Murrah buffaloes. Vet. Pract., 13: 273-275.

Lamsal D, Gautam G, Pandey SR, Shah S, Devkota B (2017). Resumption of ovarian cyclicity to induce pregnancy in anestrous buffaloes using CIDR synchronization protocol during poor breeding season. In: Proceedings of the International Buffalo Symposium 2017; Chitwan, Nepal, November 15-18, pp. 235.

Loeffler SH, De Vries MJ, Schukken YH, De Zeeuw AC, Dijkhuizen AA, De Graaf FM, Brand A (1999). Use of AI technician scores for body condition, uterine tone and uterine discharge in a model with disease and milk production parameters to predict pregnancy risk at first AI in Holstein dairy cows. Theriogenology, 51(7): 1267-1284. https://doi.org/10.1016/S0093-691X(99)00071-0

Madureira AML, Polsky LB, Burnett TA, Silper BF, Soriano S, Sica AF, Cerri RLA (2019). Intensity of estrus following an estradiol-progesterone-based ovulation synchronization protocol influences fertility outcomes. J. Dairy Sci., 102(4): 3598-3608. https://doi.org/10.3168/jds.2018-15129

Madureira AM, Burnett TA, Borchardt S, Heuwieser W, Baes CF, Vasconcelos JL, Cerri RL (2021). Plasma concentrations of progesterone in the preceding estrous cycle are associated with the intensity of estrus and fertility of Holstein cows. PLoS One, 16(8): e0248453. https://doi.org/10.1371/journal.pone.0248453

Malik RK, Singh P, Singh IJ, Sharma RK, Phulia SK, Tuli RK, Chandolia RK (2011). Ovarian response and fertility of Ovsynch-treated postpartum anestrus Murrah buffaloes. Buff. Bull., 30(4): 272-276.

MoALD (2022). Statistical information on nepalese agriculture 2020/21. Ministry of Agriculture and Livestock Development, Government of Nepal, Kathmandu, Nepal.

Moreira F, De la Sota RL, Diaz T, Thatcher WW (2000). Effect of day of the estrous cycle at the initiation of a timed artificial insemination protocol on reproductive responses in dairy heifers. J. Anim. Sci., 78(6): 1568-1576. https://doi.org/10.2527/2000.7861568x

Nanda AS, Nakao T (2003). Role of buffalo in the socioeconomic development of rural Asia: Current status and future prospectus. Anim. Sci. J., 74(6): 443-455. https://doi.org/10.1046/j.1344-3941.2003.00138.x

Nanda AS, Brar PS, Prabhakar S (2003). Enhancing reproductive performance in dairy buffalo: Major constraints and achievements. Reprod. Suppl., 61: 27- 36.

Naseer Z, Ahmad E, Singh J, Ahmad N (2011). Fertility following CIDR based synchronization regimens in anoestrous Nili‐Ravi buffaloes. Reprod. Domest. Anim., 46(5): 814-817. https://doi.org/10.1111/j.1439-0531.2010.01746.x

Neglia G, Gasparrini B, Di Palo R, De Rosa C, Zicarelli L, Campanile G (2003). Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows. Theriogenology, 60(1): 125-133. https://doi.org/10.1016/S0093-691X(02)01328-6

Nowicki A, Barański W, Baryczka A, Janowski T (2017). OvSynch protocol and its modifications in the reproduction management of dairy cattle herds an update. J. Vet. Res., 61(3): 329-336. https://doi.org/10.1515/jvetres-2017-0043

Paul V, Prakash BS (2005). Efficacy of the Ovsynch protocol for synchronization of ovulation and fixed-time artificial insemination in Murrah buffaloes (Bubalus bubalis). Theriogenology, 64(5): 1049-1060. https://doi.org/10.1016/j.theriogenology.2005.02.004

Pereira MHC, Wiltbank MC, Vasconcelos JLM (2016). Expression of estrus improves fertility and decreases pregnancy losses in lactating dairy cows that receive artificial insemination or embryo transfer. J. Dairy Sci., 99(3): 2237-2247. https://doi.org/10.3168/jds.2015-9903

Presicce GA, Senatore EM, Bella A, De Santis G, Barile VL, De Mauro GJ, Parmeggiani A (2004). Ovarian follicular dynamics and hormonal profiles in heifer and mixed-parity Mediterranean Italian buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Theriogenology, 61(7-8): 1343-1355. https://doi.org/10.1016/j.theriogenology.2003.08.013

Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL (1997). Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J. Dairy Sci., 80(2): 295-300. https://doi.org/10.3168/jds.S0022-0302(97)75937-X

Qureshi MS, Habib G, Samad HA, Lodhi LA, Usmani RH (1999). Study on factors leading to seasonality of reproduction in dairy buffaloes II. Non-nutritional factors. Asian-Austral. J. Anim. Sci., 12(7): 1025-1030. https://doi.org/10.5713/ajas.1999.1025

Sah SK, Nakao T (2010). A clinical study of anestrus buffaloes in southern Nepal. J. Reprod. Dev., 56: 208–211. https://doi.org/10.1262/jrd.09-137T

Shah SNH, Willemse AH, Van de Wiel DFM (1990). Descriptive epidemiology and treatment of postpartum anestrus in dairy buffalo under small farm conditions. Theriogenology, 33(6): 1333-1345. https://doi.org/10.1016/0093-691X(90)90051-T

Shah S, Gautam G, Kharel CN, Lamsal D, Pandeya Y, Devkota B (2017). Response of novel hormonal protocol in anestrus buffaloes during different breeding seasons. In: Proceedings of International Buffalo Symposium 2017, Chitwan, Nepal, November 15-18, pp. 202-207.

Singh J, Nanda AS, Adams GP (2000). The reproductive pattern and efficiency of female buffaloes. Anim. Reprod. Sci., 60–61: 593–604. https://doi.org/10.1016/S0378-4320(00)00109-3

Souza AH, Guemen AHMET, Silva EPB, Cunha AP, Guenther JN, Peto CM, Caraviello DZ, Wiltbank MC (2007). Supplementation with estradiol-17β before the last gonadotropin-releasing hormone injection of the Ovsynch protocol in lactating dairy cows. J. Dairy Sci., 90(10): 4623-4634. https://doi.org/10.3168/jds.2007-0172

Stevenson JS, Pulley SL, Jr Mellieon HI (2012). Prostaglandin F2α and gonadotropin releasing hormone administration improve progesterone status, luteal number, and proportion of ovular and anovular dairy cows with corpora lutea before a timed artificial insemination program. J. Dairy Sci., 95: 1831-1844. https://doi.org/10.3168/jds.2011-4767

Stevenson JS, Pursley JR, Garverick HA, Fricke PM, Kesler DJ, Ottobre JS, Wiltbank MC (2006). Treatment of cycling and non-cycling lactating dairy cows with progesterone during Ovsynch. J. Dairy Sci., 89(7): 2567-2578. https://doi.org/10.3168/jds.S0022-0302(06)72333-5

van Eerdenburg FJ, Daemen IA, van der Beek EM, van Leeuwen FW (2000). Changes in estrogen-α receptor immunoreactivity during the estrous cycle in lactating dairy cattle. Brain Res., 880(1-2): 219-223. https://doi.org/10.1016/S0006-8993(00)02870-5

Warriach HM, Channa AA, Ahmad N (2008). Effect of oestrus synchronization methods on oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes. Anim. Reprod. Sci., 107(1-2): 62-67. https://doi.org/10.1016/j.anireprosci.2007.06.007

Warriach HM, McGill DM, Bush RD, Wynn PC, Chohan KR (2015). A review of recent developments in buffalo reproduction. A review. Asian-Austral. J. Anim. Sci., 28(3): 451. https://doi.org/10.5713/ajas.14.0259

Woelders H, Van der Lende T, Kommadath A, Te Pas MFW, Smits MA, Kaal LMTE (2014). Central genomic regulation of the expression of oestrous behaviour in dairy cows: A review. Animal, 8(5): 754-764. https://doi.org/10.1017/S1751731114000342

Yendraliza BP, Zefsin BP, Udin ZJ, Arman C (2015). Estrus synchronization in swamp buffaloes. Int. Vet. Inf. Ser., Ithaca NY.

Zaabel SM, Hegab AO, Montasser AE, El-Sheikh H (2009). Reproductive performance of anestrous buffaloes treated with CIDR. Animal Reproduction, 2009; 6(3): 460-464. https://www.animal-reproduction.org/article/5b5a6071f7783717068b4778.

Zerbe H, Gregory C, Grunert E (1999). Zur Behandlung ovariell bedingter Zyklusstorungen beim Milchrind mit Progesteron-abgebenden Vorrichtungen (in German). Tierarztl Umsch, 54: 189-192.

Zerbe H, Gregory C, Grunert E (1999). Zur Behandlung ovariell bedingter Zyklusstorungen beim Milchrind mit Progesteron-abgebenden Vorrichtungen (in German). Tierarztl Umsch, 54: 189-192.

Zicarelli L (2007). Can we consider buffalo a non-precocious and hypofertile species? Ital. J. Anim. Sci., 6: 143-154. https://doi.org/10.4081/ijas.2007.s2.143