Effect of lecirelin acetate , hCG or progesterone administration on day 7 post-insemination on conception rate and progesterone concentration in cross-bred cattle

The present study was designed to evaluate the effect of administration of lecirelin acetate, hCG and progesterone after AI on ovarian picture, serum progesterone concentrations and first service conception rate in cross-bred cattle. A total of 160 lactating cross bred (Friesian x Sahiwal) cattle were divided into 4 groups after AI. The groups were treated as follows: control (injected i.m with normal saline 2ml, n=40), d 7-LA (injected i.m with lecirelin acetate100 μg, n=40), d 7-hCG (injected with hCG 3300 IU, n=40) and d 7-P4 (injected i.m with Progesterone 0.5 mg daily for 4 days, n=40) group. The hormonal treatments were given to animals on day 7 after AI. The ultrasonography and blood sampling was done before treatment and then 7 days later. All animals were examined for pregnancy through ultrasonography at 40 ± 1 day after AI. The diameter of SCL on 7 days after treatment was higher (P < 0.01) in group d 7-LA and d 7-hCG group cows as compared to control cows. In contrast, the diameter of SCL in d 7P4 treated cows did not differ from control cows. Formation of ACL was observed only in 50 % & 80 % cows in d 7-LA and d 7-hCG group respectively. No ACL was observed in control and d 7P4 cattle on day 7 after treatment. The P4 concentrations were significantly higher (P < 0.01) in d 7-hCG treated cattle as compared to control at 7 days after treatment. As compared to 10 percent First Service Conception Rate (FSCR) in controls, an increase (P < 0.05) of 50 percent and 40 percent was observed in each treatment group (d 7-hCG & d 7-LA), non-significant (P > 0.05) difference in FSCR was observed between control and d 7-P4 group. It was concluded that the use of hCG or LA, 7 days after AI is a beneficial tool to improve conception rate in cross-bred cattle whereas use of parental P4 post-breeding has no effect on SCL diameter and conception rate.


Introduction
Efficient reproduction is important for the sustainable improvement of animal productivity and is a critical factor influencing the economic viability of livestock farmers.In developing countries, lower fertility is an important factor affecting the livestock production (1).
Conception failure is related with less than normal concentrations of progesterone (P 4 ) as early as day 6 after insemination (2).Embryo development is associated with concentrations of progesterone and the ability of the conceptus to secrete the anti luteolytic hormone, interferontau (3).Exogenous P 4 has been shown to stimulate embryo development (4).A number of treatments could be used to increase peripheral P 4 concentrations after artificial insemination (AI), that improve the function of corpus luteum (CL), or supplement progestin.
A variety of hormonal regimens have been used to improve conception rates in cattle.Modern approaches have been focused on gonadotropin releasing hormone (GnRH) and human chorionic gonadotropin (hCG) hormones.But, their effects on conception rates are variable.Some authors reported increased conception rate in lactating dairy cattle (5)(6), whereas, others have reported no effect (7)(8).
Use of GnRH or its agonists (lecirelin acetate) to enhance conception rates is based on an understanding of GnRH-induced biological effects on the reproductiveendocrine system.This consequence has been brought through GnRH-stimulated LH surge and stimulating corpus luteum for P 4 production (9).The use of GnRH (10)(11) or hCG (12)(13) after AI increases P 4 secretion due to the luteotropic effect (13)(14) and/or induction of an accessory corpus luteum (11)(12)(13)(14)(15) thus, increasing P 4 concentration and, possibly, also conception.The use of P 4 releasing intravaginal devices (PRID) and controlled internal drug release (CIDR) between day 6 and 12 improves conception rate has been well documented in literature (16)(17).The parental use of P 4 after 7 days of AI has not been studied.The present study was, therefore, designed to determine the effect of lecirelin acetate, hCG and P 4 administration postbreeding in crossbred cattle.

Materials and methods
The research was conducted on 160 lactating cross bred (Friesian x Sahiwal) cows in Pakistan at a cattle farm located between Latitude 30.1984 0 N and Longitude 71.4687 0 E. The animals were in 3rd lactation and body weight of animals ranged from 312±1.70 to 318±1.94 kg and milk production ranged from 7.1±0.29 to 7.6±0.17liters per day.Each animal was fed cotton seed cake (1.6 kg), wheat bran (1.59 kg), wheat straw (4.55 kg) and green fodder (9.09 kg) daily.All cows included in the study were in their first AI postpartum and interval from calving to treatment was above 90 days and cows were free from brucellosis, tuberculosis and mastitis.Each cow was identified by tattoo in the ear.The animals had free access to drinking water.Semen of single bull of Sahiwal breed was used for AI.All the inseminations were done by single technician.Following AI, the animals were randomly divided into four groups viz., control (normal saline 2ml, n=40), d 7-LA (lecirelin acetate100 µg, Dalmeralin; Fatro, Itlay, n=40), d 7-hCG (hCG 3300 IU, Choriomon; Lugano, Switzerland, n=40) and d 7-P 4 (Progesterone 0.5 mg daily for 4 days, Pregtone; Selmore Pharmaceuticals, Pakistan, n=40) group.The hormonal treatments were given i.m to animals on day 7 after AI.
Ovaries of all experimental animals were scanned through trans-rectal probe (7.5 -MHz, Honda, Japan).The ultrasonography was done before treatment and then 7 days later.Blood sampling from all experimental cows was done before hormonal treatment and 7 days after treatment.Blood was collected from jugular vein with sterilized disposable syringe with 18G needle.The blood samples were brought to the lab in cooled box.Serum was separated by centrifugation at 3000 rpm for 15 minutes.The serum samples were stored at -20˚C till analysis (18).Progesterone concentrations were measured through by using a commercially available kit (Bio Check, Inc, Lot.RN-36893).The sensitivity of assay was 0.2 ng/ml, whereas inter and intra-assay coefficients of variation were < 15 %.All cows were examined for pregnancy through ultrasonography at 40 ± 1 day after AI.The data regarding first service conception rate (FSCR) were analyzed by Chi square test, whereas diameter of spontaneous corpus luteum (SCL), accessory corpus luteum (ACL) and P 4 concentrations were analyzed by ANOVA (19).

Results
The ovarian response in cross-bred cows for different experimental groups is summarized in Table-1.The diameter of SCL on 7 days after treatment was higher (P<0.01) in d 7-LA and d 7-hCG group as compared to control.In contrast, the diameter of SCL in d 7-P 4 did not differ from control.Since the purpose for the use of lacerilin acetate and hCG was to induce ACL during first wave of follicles at day 7 after AI. 12.8 ± 0.5 -12. 9 ± 0.5 -13.8 ± 0.4* 9.2 ± 0.3 a 14.1 ± 0.4* 11.6 ± 0.4 a * a There was significant difference (P < 0.01) Serum progesterone (P 4 ) concentrations were determined in all experimental cows at the time of treatment and 7 days later (Table -2).In all experimental groups, the serum P 4 concentrations were increased (P<0.01)7 days after treatment as compared with the day of treatment (Fig. 1).The P 4 concentrations were significantly higher (P<0.01) in d 7-hCG and d7-LA cattle as compared to control at 7 days after treatment.As compared to 10 percent FSCR in controls, an increase (P<0.05) of 50 percent and 40 percent was observed in each treatment group (d 7-hCG & d 7-LA).Non-significant (P>0.05)difference in FSCR was observed between control and d 7-P 4 group.

Discussion
In the present study, cross-bred cattle were treated with LA, hCG or P 4 , 7 days after AI to induce ACL and increase circulating progesterone concentrations.About 50 % & 80 % cows develop ACL in response to LA and hCG.Development of ACL during this phase of estrous cycle is in agreement with other studies for GnRH (20) and hCG (13)(14)(15)(16)(17)(18)(19)(20)(21).The increase in size of SCL in response hCG is attributed to the fact that hCG functions like LH. hCG binds with LH receptors at target site and affects on small luteal cells to secrete P 4 .Size of large luteal cells increased when hCG was given on day 3-5 of ovine estrous cycle (22).Since about 80 % P 4 is synthesized from large luteal cells (22), therefore, in our study increase in diameter of SCL was due to increase in size of large lueal cells.Higher P 4 in hCG treated cattle in our study is due to the effect of hCG on SCL as well as ACL.
Formation of ACL in response to LA is reported in another study also (21).In our study, P 4 concentrations were higher in LA treated cattle than control and are in agreement with Howard et al. (23).The results of LA and hCG are comparable for development of ACL in our study, LA and hCG were equally effective in inducing CL when given on day 6 of estrous cycle in heifers (11).In our study, we replaced the use of CIDR in previous studies with daily P 4 administration for 4 days.Administration of P 4 (i.m) for 4 days did not increased the SCL diameter as well as serum P 4 concentrations as compared with control group.
It has been reported that administration of hCG/ GnRH in early luteal phase between 4-6 day of estrous cycle induced ovulation of the first wave dominant follicle and formation of accessory CL.Higher FSCR was observed in hCG and LA cattle.These findings are in agreement with Santos et al. (13) who evaluated the effects of hCG administered on day 5 after AI on CL number, P 4 concentrations, conception rate and pregnancy loss in highproducing dairy cows.The findings of present study also corroborate with Stevenson et al. (21).Daily P 4 administration did not affected FSCR in present study.The P 4 concentration during luteal phase after AI is associated with good quality embryo development (24).During the process of maternal recognition of pregnancy, high quality embryo will suppress luetlytic process through IFN.τ (25)(26).In our study, beneficial effect of hCG and LA was observed through improvement of SCL diameter, development of ACL and thus higher circulating P 4 concentrations.It was concluded that use of hCG or LA, 7 days after AI is a beneficial tool to improve conception rate in cross-bred cattle.The use of parental P 4 post-breeding has no effect on SCL diameter and conception rate.

Fig. 1 :Table 2 :
Difference in serum progesterone concentrations before treatment and 7 days later in experimental cross-bred cattle.Serum progesterone concentrations and first service conception rate in cross-bred cattle treated with LA, hCG and progesterone on day 7 after AI Progesterone Concentration (ng/ml) after treatment 3.4 ± 0.3 3.6 ± 0.3 4.7 ± 0.3* 5.1 ± 0.2* First Service Conception Rate (%) 10 10 40* 50* *There was significant difference (P < 0.01) Effect of lecirelin acetate, hCG or progesterone administration on day 7 post-insemination on conception rate and progesterone concentration in cross-bred cattle

Table 1 :
Formation of ACL was observed only in 50 % & 80 % cows in d 7-LA and d 7-hCG group respectively.ACL diameter was greater in d 7-hCG cattle in comparison to d 7-LA cattle.No ACL was observed in control and d 7-P 4 cattle on day 7 after treatment.Ovarian response in cross-bred cattle treated with LA, hCG and progesterone on day 7 after AI: (SCL and ACL diameter: mm)