Effect of biostimulation and estrus synchronization on estrus response and fertility rate in primiparous and multiparous Kundhi buffaloes

Anestrus and unobserved estrus are one of the crucial managemental lapses in the livestock business. This research was planned to assess the efficiency of different estrus synchronization procedures with bull exposed and with non-bull exposed groups in primiparous and multiparous buffaloes at Kundhi buffalo farm Rorhi and its surroundings. Total 40 (n=20 primiparous, n=20 multiparous) buffaloes were used in this study. The chosen buffaloes were divided into two main groups, A (BE, Bull-Exposed) and group B (NE, Non-Exposed). Based on the treatments A group was further sub-divided into two sub-groups, OvSynch bull-exposed (OBE) A1 and prostaglandin bull exposed (PBE) A2. Also, B group was parted into dual sub-groups Ovsynch non-exposed (ONE) and prostaglandin non-exposed (PNE) group B2. Results of the present study showed that estrus response differed significantly among the primiparous and multiparous Kundhi buffalo in all groups (P<0.05). The overall estrus response (50% and 100%) and pregnancy rates (40% and 70%) were higher in both primiparous and multiparous animals of BE groups as compared to primiparous (60% and 70%), and multiparous animals (30% and 60%) of NE groups. The overall estrus, duration was also higher in the primiparous (22 ± 0.66 hours) and multiparous animals (20 ± 2.05 hours) of BE groups, in contrast to 16 ± 0.95 hours and 14 ± 0.84 hours of NE groups. These findings suggested that the use of bull with OvSynch protocol it could efficiently be used to improve the estrus incidence and pregnancy rate in primiparous and multiparous buffaloes as compararion double prostaglandin protocol.


Introduction
Reproductive performance of an animal is a crucial factor in the livestock business.There are many factors which hinder the reproductive performance of an animal.Amongst these, anestrus or unobserved estrus is the prominent one.In anestrus, there is complete sexual inactivity with no manifestation of heat.The ovary becomes inactive and normal cyclic activities are seen to be immobile in this condition.There are several causes of anestrus, e.g., hormonal imbalance, poor nutrition, acquired abnormalities of the female reproductive system and seasonal changes [1].Estrus Synchronization (ES) is one of the solutions to overcome these problems.Reproductive performance of female animals can be enhanced by the use of ES.Numerous ES techniques have been developed previosly in animals for the improvement of estrus response and fertility rate [2-4].In these ES practices progesterone, prostaglandin, GnRH, and estrogens alone or in numerous combinations are used [5,6].ES involves manipulating the estrous cycle to cause as many females as possible to enter estrus at a specific time.The first efforts to do so began in the late 1960s by administering oral progesterone and an estrogen injection [7].Since then numerous estrous synchronization protocols are continued to be developed to facilitate the usage of artificial insemination and for the improvement of the reproductive proficiency of farm animals.Estrus Synchronization and artificial insemination are two of the most significant management practices available to producers to increase the reproductive performance of their animals [8].Bio stimulation may be defined as a male stimulatory effect on estrus response and ovulation rate in the female via pheromones [9].Pheromones are defined as airborne chemicals released from the feces, cutaneous glands or from urine, that are sensed by the respiratory or olfactory systems that cause endocrine response and behavioral changes [10].In male animals, pheromones are largely released from urine and they affect on hypothalamic-pituitary-adrenal (HPA) activity of female animals [11,12].It has been stated by exposing the cows to bulls (biostimulation) before, during, and after a GnRH-based ES protocol following fixed timed artificial insemination improved pregnancy rate in bovine animals [13,14].However, information related to the effects of bull exposure combination with estrus synchronization in primiparous and multiparous Kundhi buffaloes is scarce.Therefore, the current research was established, with the goal to evaluate the biostimulatory effect of bulls to improve the estrus response & fixed-timed artificial insemination pregnancy rate in primiparous and multiparous Kundhi buffaloes using different estrous synchronization protocols.

Animals and their management
Total 40 (n=20 primiparous, n=20 multiparous) buffaloes were used in this study.These animals were raised on semi intensive managemental circumstances at Kundhi Farm Rohri and its surroundings.Before the start of experiment all the buffaloes were confirmed nonpregnant by rectal palpation.

Experimental design
The chosen buffaloes were divided into two main groups, A (BE, Bull-Exposed) and group B (NE, Non-Exposed).Based on the treatments A group was further sub-divided into two sub-groups, OvSynch bull-exposed (OBE) A1 and in prostaglandin bull exposed (PBE) A2.Similarly, B group was parted into two sub-groups ovSynch non-exposed (ONE) and prostaglandin non-bull exposed (PNE) group B2.

Sub-group-A-1.OvSynch Bull Exposed (OBE) group n=10
Estrus Synchronization was implemented with the OvSynch protocol, in which 02 ml GnRH on day 0 and 9th and 05ml PGF2α was injected on day 7, and also these buffaloes were exposed to an aproned bull two times a day i.e. at 6 am and 6 pm for half an hour, from the starting of experiment to artificial insemination.

Sub-group-A2. Prostaglandin F2α Bull-Exposed (PBE) group n=10
In this group double PGF2α was injected at the interval of 11-days.After injection of PGF2α, these animals were also exposed to an intact, aproned bull for 30 minutes, two times in a day (6am, and 6pm) from the starting of treatment till artificial insemination.

Sub-group-B-1.OvSynch Non Bull Exposed (ONE) group n=10
Synchronization was performed in these animals with a similar procedure, as used in the animals of group A-1.But the animals of this group were not exposed to bull from the start of experiment till artificial insemination.

Sub-group-B-2.Prostaglandin F2α Non-Exposed (PNE) group n=10
The treated animals were not exposed to the bull, but synchronization was performed with a similar method, as recived by subgroup B2 animals.

Estrus detection
Group A-1 and B-1 animals were observed after the injection of 2 nd GnRH for behavioral changes to confirm the heat.However, animals of group A-2 and B-2 were watched closely after 2 nd PGF2α inoculation to check the estrus.In group A1 and B1 artificial insemination was performed two times i.e. 12 and, 24 hours after the last gonadotropin inoculation.While artificial insemination was done after 72 and 96 hours in A2 and B2 group, following second prostaglandin shot by frozen thawed semen got from Directorate of Animal Breeding, Livestock Department Government of Sindh.

Statistical analysis
Results were calculated by using graph pad instate 3.05 versions of the statistics through Chi square test and ANOVA.The significant was considered to be at P<0.05.

Results
The current research was carried out to evaluate with or without biostimulation the effectiveness of different protocols of estrus synchronization (ES), in primiparous and multiparous Kundhi buffaloes.The outcomes are existing in the following tables.

Occurrence of estrus
The effect of estrus synchronisation with and without bull exposure on the appearance of estrus in OvSynch and double prostaglandin F2α groups in different parities of Kundhi buffaloes are defined in (Table 1).Intensity of estrus significantly (P<0.05)differed among the primiparous and multiparous Kundhi buffaloes in all groups.Furthermore, higher estrus response (100%) was detected in all multiparous animals, while primiparious animals showed (60%) and (40%) in OBE and ONE group respectively, Likewise, OvSynch non-exposed treatment induces higher estrus response in multiparous animals as compare to primiparous respectively.Overall estrus response (Table 2) was observed higher in the primiparous and multiparous animals of BE, (50%), (100%), groups as compared to NE (60%), (70%) groups respectively.

Pregnancy rate
The results of synchronization of estrus with bull exposed and non bull exposed on pregnancy rate following OvSynch and double prostaglandin F2α in primiparous and multiparous Kundhi buffaloes are precised in (Table 5).These results revealed that pregnancy rate significantly (P<0.05)vary amongst all the groups.While overall pregnancy rate was greater in the primiparous (40%) and multiparous (70%) animals of bull exposed (BE) group as compared to nonexposed (NE) (30%) & (60%) groups (Table 6).Furthermore, higher pregnancy rate was identified in the primiparous and multiparous animals of OvSynch bull exposed group (40%) & (80%) as compared to prostaglandin F2α bull exposed (40%),(60%) group.However in primiparous and multiparous animals of OvSynch non-exposed (40%), (60%) and prostaglandin F2α non-bull exposed group were (20%) & (60%) respectively.In both cases estrus response and estrus duration, the effect may also be differed due to the difference in the species.Cows respond to treatment might be in a different manner as compared to buffaloes.Estrus duration was higher in the primiparous and multiparous animals of both treated bullexposed groups as compared to non-exposed groups.These results are in agreement with Khanh et al. [11] for the primiparous cows but not for multiparous.However, in another study by Flores et al. [18] who reported that reported the primiparous cows represent low estrus duration as compared to multiparous cows.The difference observed between the present study, and the above mentioned two studies may be due to the different breeds and environment.In different environmental condition and in the breeds the esrus response varies.In current findings, duration of estrus was apparent in primiparous (22±0.663hours) and multiparous (20 ± 2.05 hours) animals of bull-exposed group followed by primiparous ((16 ± 0.954 hours) and multiparous (14 ± 0.841 hours) animals nonbull exposed group respectively.Our results of estrus duration are greater than those stated by Roeflos et al. [19] who declared that duration of estrus in primiparous is 13.6 hours and in multiparous animals 10.8 hours.These alterations in findings may be due to different environmental conditions, type of housing, how estrus was detected, and the frequency of handling.In all groups, a significantly higher pregnancy rate was noted in multiparous animals as compared to primiparous animals.The present findings are similar to the Khanh et al. [11] who observed a higher pregnancy rate in multiparous animals as compared to primiparous animals.But they reported the non-significant difference in pregnancy rate among the primiparous and multiparous cows.Murugavel et al. [20] reported 57.6% conception rate in multiparous cows and 40.6% in primiparous with progesterone based estrus synchronization treatment.While in current study high conception rate was observed in multiparous buffaloes (80%) and (60%), while in primiparous (40%) conception rate was observed in case of OvSynch bull exposed and prostaglandin F2α bull exposed group respectively.

Conclusion
This study concluded that the use of bull with OvSynch protocol of estrus synchronization could efficiently be used to improve the response of estrus and fertility rate in primiparous and multiparous Kundhi buffaloes as compare to prostaglandin group.

Authors' contributions
Dissimilar superscript within the similar column represents significant results, P= 0.049