Improvement of Fertility Rate Using Ovsynch and Biostimulation Protocols with Timed Artificial Insemination in Thari Cattle

Ghulam Nabi Dahiri1, Akeel Ahmed Memon2, Qudratullah Kalwar3*, Asmatullah Kaka2, Waseem Ali Vistro4, Yar Muhammad Jalbani3, Kiran Nazish3 and Jai Parkash Kolhi5 1Directorate of Animal Breeding, Hyderabad 2Department of Animal Reproduction, Sindh Agriculture University, Tandojam 3Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand, Sindh 4Department of Anatomy and Histology, Sindh Agriculture University, Tandojam 5Department of Animal Products Technology, Sindh Agriculture University, Tandojam

The study was carried out to compare efficacy of estrus synchronization protocols with or without biostimulation in Thari cattle. A total of forty Thari cattle maintained at semi intensive management conditions at Thari Cattle Farm Nabisir Road, Distt: Umerkot were used in the study. The selected animals were divided into four groups to observe estrus signs and artificial insemination. Animals of group B and D were treated with OvSynch (GnRH at day 0 followed by PGF2α day 7 and 2nd GnRH at day 9) for estrus synchronization but D was bio-stimulated and B was not bio-stimulated. Animals in group A and C were treated with 2ml normal saline on day 0, 7 and 9 of the treatment. Group C was biostimulated and A was not biostimulated. The results showed that estrus response (60 %) and pregnancy rate (50%) was higher, onset of estrus was earlier (54.89 ± 1.20), duration of estrus was long (23.17 ± 1.20) in group D as compare to other groups. It was concluded in the present study that the biostimulation with ovsynch protocol can effectively be used to induce cyclic activities and increase estrus response and fertility rate in Thari cattle. R eproductive efficiency of dairy animals has been decreasing in the past 50 years. Reduced reproductive efficiency not only frustrate the dairy producers and their consultants but also substantially reduce the dairy farm profitability. Several approaches have been applied to improve reproductive efficiency of dairy animals and it has been found that the use of hormones for induction of estrus in anestrus and/or post-parturient animals effectively reduce calving interval thus improving reproductive performance Tenhagen et al., 2005). Induction of estrus in a group of animals or bringing a group of females into estrus (heat) at a predetermined time with the use of hormones is called estrus synchronization (ES).
ES involves the manipulation of the estrus cycle in order to cause as many females as possible to enter estrus at a specific time. The first efforts to do so began in the late * Corresponding author: qudratullahkalwar@gmail.com 0030-9923/2022/0001-0001 $ 9.00/0 Copyright 2022 Zoological Society of Pakistan 1960's by administering oral progestins and an estrogen injection (Wiltbank and Kasson, 1968). Since then numerous ES protocols continue to be developed in order to facilitate the use of artificial insemination (AI) and improve the reproductive efficiency of farm animals. Utilizing AI and ES offers many benefits to farmers. AI allows access to elite genetics that would not otherwise be available for use. Using AI in combination with ES can improve the reproductive efficiency and management. ES and AI are two most important management practices available for producers to increase reproductive performance of their animals (Jabeen et al., 2012;Roy and Prakash, 2009).
Furthermore, it has been reported that fixed-timed AI pregnancy rate and overall AI pregnancy rate of bovine animals improved by exposing the cattle to bulls (biostimulation) before, during, and after a GnRH-based ES protocol Tauck and Berardinelli, 2007). Biostimulation may be defined as the stimulatory effect of a male on estrus and ovulation through genital stimulation, olfactory pheromones, or other less well O n l i n e

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defined external cues such as tactile, visual and auditory (Chenoweth, 1983). However, very little information is available on the combined use of biostimulation with ES protocols in Thari cows. Hence, this study was intended with the aim to determine the biostimulatory effect for improvement of the synchronization of estrus and fixedtimed AI, pregnancy rates of Thari cows, with use of Ovysynch synchronization protocol.

Materials and methods
Forty Thari cattle of 1 st to 4 th parity maintained on semi intensive management conditions at Thari Cattle Farm Nabisir Road, Distt. Umerkot and its surroundings were used in the study. Before the start of experiment rectal palpation was done to confirm non-pregnant females. The feeding and drinking was done according to routine practices of the farm.
The females were grouped into four groups each of 10: (i) Group-A (non-treated without biostimulation) in which animals received 2ml sodium chloride (Nacl) on 0, 7 and 9 th of the experiment and were not exposed to bull. (ii) Group-B (Ovsynch without biostimulation n=10) in which animals were administered with 2ml of GnRH (Dalmarelin, Fatro-Italy) on day 0, on day 7 5ml PGF2α (Lutalyse ® , Pfizer-USA) and 2 nd injection of 2ml GnRH (Dalmarelin, Fatro-Italy) on day 9. Females were not exposed to bull. (iii) Group-C (non-treated with biostimulation) in which females were injected 2ml of normal saline on day 0, 7 and 9 of the synchronization and females were exposed to biostimulation for half an hour daily (6 am and 6 pm) from day 0 of synchronization to artificial. (iv) Group-D (Ovsynch with biostimulation) in which females were injected with Ovsynch protocol which involves administration of 2ml GnRH (Dalmarelin, Fatro-Italy) on day 0, injection of 5ml PGF2α (Lutalyse ® , Pfizer-USA) on day 7 and a 2 nd injection of 2ml GnRH (Dalmarelin, Fatro-Italy) at day 9. Females were also exposed to biostimulation for half an hour, daily (6 am and 6 pm) from day 0 of synchronization to AI.
Females of all groups were watched daily for heat from the start of the treatment (day 0). All females were inseminated after the last injection (day 9) following protocols of injection at 12 and 24 h after onset of estrus with semen received from Directorate of Animal Breeding (DAB), The following parameters were recorded in all groups. (i) Onset of estrus were watched closely (in females of all groups) after 2 nd injection of PGF2α for behavioral signs of heat. The major bevioral signs were observed including mounting, bellowing, sniffing, restlessness and micturition. The animals were inseminated artificially at 12 and 24 h, after third injection of GnRH in group D and B. However, females of A and C group were artificially inseminated twice 72 and 96 h after the PGF 2α injection.
(ii) Duration of estrus was recorded by observing estrus signs visually from onset of estrus signs till cessation of estrus signs. (iii) Fertility/ Pregnancy rate was determined by per rectum palpation at 2 months post AI.
Statistical package Graph pad instate 3.05 versions of was used to analysis the data. Analysis of variance (ANOVA). To determine significant Difference between means of groups' chi square test was used. Significant level was considered at P<0.05.

Results
The effect of ovsynch protocol with or without biostimulation on estrus response, onset of estrus, estrus duration and fertility rate of Thari cattle is shown in Table  I. Estrus response was significantly different among the groups (P<0.05). Ovsynch bull-exposed group showed higher estrus response as compared to other groups. In addition to that multiparous animals significantly showed better estrus response as compared to primiparous Thari cattle in all groups (Table I). The onset of estrus was earlier with ovsynch biostimulated group than non-treated non-exposed, and control group. Similarly onset of estrus was lower in than ovsynch bostimulated group (54.89± 1.20) and higher in control group (60 ± 0.00), respectively (Table I). Moreover, onset of estrus differ significantly among bull (exposed and non-exposed) group (P<0.05) and primiparous animals showed earlier onset of estrus as compare to multiparous animals ( Table I). The estrus duration did not differ significantly among ovsynch bull exposed and non-exposed the group (P<0.05). While duration of estrus was higher with ovsynch bostimulated group than non-treated non-exposed, and control groups. However, primiparous animals showed longer estrus duration as compare to multiparous animals (Table I). Pregnancy rate significantly differed among the groups (P<0.05). Ovsynch bull-exposed group showed higher pregnancy rate as compare to other treated and control groups (Table I). In addition to that multiparous animals showed significantly higher pregnancy rate as compare to primiparous Thari cattle in all groups (Table I).

Discussion
ES technique is applied to reduce the problems of silent heat and anestrus probloms in cattle. Hormones like gonadotropin releasing hormone prostaglandin F2α and analogues are being used for ES (Khumran et al., 2012). The ovsynch synchronization protocol is the most popular ES protocol which consists of series of GnRH -PGF2α-GnRH injection treatments since last decade and provides satisfactory fertilization to timed AI (Jabeen et al.,2012). This protocol is being used in buffaloes with satisfactory pregnancy rates (Chaikhun et al., 2010). Biostimulation or effect of a male via genital stimulation, olfactory pheromones, or other less defined external sign such as tactile, visual and auditory have been used to induce estrus (Chenoweth, 1983). In the current study response of estrus was significant in the cattle with ovsynch and biostimulated group than non-treated non-exposed, and control groups. Roy and Prakash (2009) reported 18% reduction in anestrous rate with synchronization and biostimulation. Estrus response was higher with ovsynch with biotimulation (60%) than ovsynch non-exposed to biostimuation (50%). However, difference was nonsignificant among groups (P>0.05). These results are in agreement with the findings of Berardinelli et al. (2001). They reported that the response of estrus was more in postpartum cattle exposed to biostimuation during synchronization of estrus than non biostimulated. Ahmed et al. (2011) reported 90% of estrus response in cattle exposed to biostimulation than 65% females without biostimulation. Similarly, Alberio et al. (1987) also reported 67.9% estrus response with biostimulation than 32.7% without biostimulation.

O n l i n e F i r s t A r t i c l e
In the current study multiparous animals showed significantly higher estrus response as compared to primiparous Thari Cattle in all groups. All multiparous animals showed significantly better estrus responses as compared to primiparous animals among the groups (P<0.05). Contrary to this, Khanh et al. (2012) observed non-significant difference among the primiparous and multiparous cattle, however, estrus response was higher in multiparous than primiparous animals. Similarly, Ghosh et al. (2012) reported lower estrus response in primiparous (38.9%) than in multiparous (77.7%) cattle. Number of factors could be affecting this outcome such as synchronization protocols may have induced difference in physiological response and played a major role in variation among observations. In the present study ovsynch method of ES were used whereas Khanh et al. (2012) used progesterone supplementation in the form of controlled internal drug releasing device (CIDR) for ES. In this study estrus duration was higher with ovsynch biostimualtion than non-treated without biostimualtion, and control groups. This finding lies in range with Khanh et al. (2012). However, Flores et al. (2006) observed higher duration of estrus (P<0.05) in multiparous than primiparous cattle. The difference observed between present study and above two studies may be due different breeds and environmental condition which induce variation response among synchronization treatments. In the current study the duration of estrus were 23 and 22 h in primiparous and multiparous animals, respectively. This was higher than results by Roelofs et al. (2005). They reported 13.6 and 10.8 h duration of estrus in primiparous and multiparous females, respectively. The difference in results may be due to difference in housing, handling and environmental condition.
In this current study pregnancy rate was significantly higher in ovsynch with biostimualations group 50% as compared to ovsynch without bio-stimualations group (40%). Tauck and Berardinelli (2007) found 59% and 37% fertility rate with biostimulation and without biostimulation Improvement of Fertility Rate Using Ovsynch and Biostimulation Protocols O n l i n e

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in cattle, respectively. These findings support the result of current study. In the present study 50% and 40% pregnancy rate were recorded with biostimulation and without biostimulation. These findings are lower than those reported by Purabi et al. (2011). Ahmed et al. (2011) found 81.82%, 66.7% and 40.0%, 33.7% conception rate with biostimulation and without biostimulation group, respectively. The difference in the results may be due to difference in environment, breed and ES protocol.
In the current study 60% conception rate was observed in multiparous cattle in ovsynch with bio-stimualation and ovsynch without biostimulations, while in primiparious these values were 40% and 20%, respectively with ovsynch with biostimualations and ovsynch without biostimualation. The present results are in line with those of Khanh et al. (2012) who observed higher pregnancy rate in multiparous animals compared to primiparious animals. However, the differences were non-significant. Murugavel et al. (2009) observed 57.6% conception rate in multiparous cattle and 40.6% in primiparous cattle with progesterone based ES protocol.

Conclusions
It can be concluded from the present study that: The estrus response and pregnancy rate was higher in bull exposed (with biostimulation) groups as compare to nonbull exposed (without biostimulation) groups.

Statement of conflict of interest
The authors have declared no conflict of interest.