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The effect of different doses of Gonadorelin on ovarian follicle dynamics in river buffalo (Bubalus bubalis)
Theriogenology 62 (2004) 1283–1291
The effect of different doses of Gonadorelin on ovarian follicle dynamics in river buffalo (Bubalus bubalis) A. Rastegarniaa,1, A. Niasari-Naslajia,b,*, P. Hovareshtia, F. Sarhaddib, M. Safaeib a
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, I.R. Iran b Animal Sciences Research Institute, Karaj, I.R. Iran
Received 25 September 2003; received in revised form 18 November 2003; accepted 17 January 2004
Abstract The objective of this study was to determine the response of the ovarian dominant follicle to the different doses of GnRH in river buffalo. The estrous cycle of 12 river bufflaloes was synchronized using norgestomet implant for 12 days in association with two injections of prostaglandin F2a analogue on Days 0 and 7 of implant insertion. On Day 6 or 7 of the ensuing cycle (Day 0 of the experiment), females received a norgestomet implant in conjunction with two prostaglandin injections on Days 0 and 1. On Day 6 of the experiment, females were randomly allocated into three groups. At this time, Group 1 and 2 females were given an i.m. injection of 50 or 100 mg Gonadorelin, respectively. Group 3 females did not receive any further treatment and were considered as control. All females were given prostaglandin on Day 12 and implants were removed on Day 13 of the experiment. The results revealed that in the control group, ovarian dominant follicle became persistent throughout the experiment; whereas, the persistent dominant follicle in all females belonging to Group 2 (100 mg GnRH) and one female in Group 1 (50 mg GnRH) ovulated within 48 h, subsequent with an emergence of a new follicular wave and an increase in plasma progesterone concentration within 72 and 96 h after GnRH injection, respectively. In conclusion, 100 mg of Gonadorelin seems to be the most effective dose to induce ovulation followed by an emergence of a new follicular wave in river buffalo. # 2004 Elsevier Inc. All rights reserved. Keywords: River buffalo; GnRH; Effective dose; Dominant follicle
* Corresponding author. Tel.: þ98-21-6923510; fax: þ98-21-6933222. E-mail address: [email protected] (A. Niasari-Naslaji). 1 Present address: Azad Islamic University, Urmia, West Azerbaijan, Iran.
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.01.014
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1. Introduction GnRH and GnRH analogues have been used in river buffaloes to reduce post-partum anestrus period [2,3,17,24,28,39,41], induce ovulation at the end of superovulation programs [18,27,43] and synchronize follicular emergence and ovulation in estrus synchronization programs and fixed time insemination protocols [4,8]. Different doses of GnRH have been used in these studies; however, there is no published controlled study describing the effects of different doses of GnRH on ovarian follicle growth in cyclic river buffalo. The use of the minimum effective dose of GnRH will enhance the cost-effectiveness of reproductive programs in the buffalo industry. The objective of the present study was to determine the minimum effective dose of GnRH to control the ovarian follicle growth in this species.
2. Materials and methods 2.1. Experimental location The experiment was conducted at the Buffalo Breeding and Extension Training Center, Urmia, West Azerbaijan, Iran (Latitude: 380 2300 N; Longitude: 470 4000 E; Altitude: 1568.5 m) during March–May 2002. 2.2. Experimental animals Twelve cyclic buffaloes, 10:4 1:36 years of age, weighing 498 33:04 kg LW, 224:2 48:66 days post-partum and with regular estrous cycles were selected for this study. They received a ration of alfalfa hay and concentrate including 50% barley, 15% cotton seed meal, 33% bran, 1% salt and 1% DCP. 2.3. Experimental design The estrous cycles of females were synchronized using a norgestomet implant (3 mg,17a-acetoxy-11b-methyl-19-norpreg-4-en-3,20-dione, Crestar1, Intervet, Holland) for 12 days in association with two injections of a synthetic prostaglandin F2a analogue (PGF2a, Luprostiol, Prosolvin1, Intervet, Holland) on Days 0 and 7 of the norgestomet insertion. On Days 6–7 of the ensuing cycle (Day 0 of the experiment), each received the norgestomet implant and two consecutive injections of PGF2a on Days 0 and 1 of the experiment. On Day 6 of the experiment, they were randomly assigned into three equal groups. Those in Group 1 (n ¼ 4) and Group 2 (n ¼ 4) received 50 or 100 mg GnRH analogue, i.m. (Gonadorelin, Fertagyl1, Intervet, Holland), respectively. Those in Group 3 (n ¼ 4) were not treated with GnRH and were considered as the control group. All cows were treated with PGF2a analogue on Day 12, and norgestomet implant was removed on Day 13 of the experiment.
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2.4. Ultrasound examination Ultrasound examination of the ovarian follicles was conducted on a daily basis starting from Day 0 of the experiment to the day of ovulation. Ovarian ultrasonography was performed as previously described [29] using a real time, B-mode scanner (Pie-Medical 480, Holland) equipped with a 5 MHz linear-array rectal transducer. During each examination, drawings of the ovaries were made, recording the location and the diameter of the individually identified follicles, 4 mm in diameter [19] and corpus luteum. A follicle, 10 mm in diameter, was considered morphologically to be a dominant follicle. The day of the emergence of a new follicular wave was defined as the day that the dominant follicle was, retrospectively, identified at a diameter of 4–5 mm [15,19]. The time that the dominant follicle disappeared was considered as the day of ovulation. 2.5. Blood sampling and plasma progesterone assay Daily blood sampling for the determination of plasma progesterone concentrations was performed from Day 0 of the experiment to the day of ovulation. Blood samples were collected by jugular venipuncture into 10 ml vacutainer heparinized tubes (Pars Khavar, Iran). Collected blood samples were centrifuged immediately at 2500 rpm for 15 min and plasma samples were stored at 20 8C until hormone analyses. Plasma concentrations of progesterone were determined using an extraction, single antibody radioimmunoassay [11]. The sensitivity of assay was 0.2 ng/ml and the inter- and intra-assay coefficients of variation were 10.2% (n ¼ 8) and 8.3% (n ¼ 8), respectively. 2.6. Statistical analyses Changes in the plasma concentrations of progesterone and the diameter of ovarian follicles over time were analyzed for the effects of treatments, day and treatment by day interaction using procedure GLM in SAS/STAT [36] by either univariate or multivariate analysis [14], with repeated measures analysis included in the model. Multivariate analyses were used where variance and covariance structures over time did not conform to the analysis of variance assumptions, using sphericity test. Single-points measurements for follicular characteristics and wave emergence were compared using ANOVA followed by Tukey’s Honestly significant difference [47]. In case the assumptions of parametric tests were not achieved, the non-parametric ANOVA for a single factor (Kruskal–Wallis one-way ANOVA) of SAS/STAT was used. Data were presented as mean S:E:M:
3. Results As all experimental groups received the same treatment until Day 6 of the experiment (the day of GnRH injection), all data for this period of experiment were pooled and analyzed together.
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3.1. Plasma progesterone concentration The concentration of plasma progesterone was monitored from Day 0 (Days 6–7 of estrous cycle ¼ day of norgestomet insertion) to Day 16 of the experiment (Fig. 1). This was 1:9 0:28 ng/ml at the time of norgestomet insertion, which was reduced to a value below the sensitivity of the progesterone assay within 24 h after the first prostaglandin injection and remained unchanged until Day 6 of the experiment. In the control group females, that did not receive further treatment, the plasma progesterone concentrations remained constant at the basal concentration throughout the experiment. Following GnRH administration, in only one animal (#329) which was in the group that received 50 mg Gonadorelin, the plasma concentration of progesterone increased significantly (P < 0:05) at 96 h after GnRH injection and this trend continued until Day 12 of the experiment (day of prostaglandin injection). In all females receiving 100 mg Gonadorelin, the plasma progesterone concentration started to increase at 96 h after treatment (0:53 0:06 ng/ml; P < 0:05) and reached a concentration of 1 0:07 ng/ml (P < 0:05) at the time of PGF2a treatment (Day 12 of the experiment). It decreased to a value below the sensitivity of assay, 24 h after prostaglandin injection (Fig. 1). 3.2. Ovarian follicle dynamics On Day 0 of the experiment (Days 6–7 of the estrous cycle), 10 females had an ovarian follicle 10 mm (10:9 0:36 mm in diameter), and all of them had a corpus luteum (17:4 1:38 mm), which regressed following two injections of PGF2a on Days 0 and 1. From Days 0 to 6 (day of GnRH injection) of the experiment, the ovarian follicles progressively grew in all animals (P < 0:05; Fig. 2). At the time of GnRH treatment (Day 6), 11 cows had a dominant follicle (11:9 0:47 mm). Only one, in the group that received 50 mg of GnRH, did not have dominant follicle; therefore, it was excluded from the data set. In all animals in the control group, the ovarian follicle maintained and became ovulatory. In
Fig. 1. The plasma progesterone concentrations from Day 0 (day of norgestomet insertion) to Day 15 of the experiment in river buffaloes that received 0, 50 and 100 mg GnRH on Day 6 of the experiment.
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Fig. 2. The growth pattern of ovarian follicle from Day 0 (day of norgestomet insertion) to Day 16 of the experiment in river buffaloes that received 0, 50 and 100 mg GnRH on Day 6 of the experiment.
contrast, the dominant follicle in the group that received 100 mg GnRH ovulated and corpus luteum was detected at 72 h after GnRH injection. The diameter of the induced corpus luteum was 12:5 0:29 mm at the time of PGF2a treatment (Day 6 after GnRH injection). In this group, the emergence of a new follicular wave occurred at 72 h after GnRH injection. Ovulation of the dominant follicle in control and 100 mg GnRH groups was detected between 72 and 96 h (three females at 72 h and one female at 96 h) after norgestomet withdrawal (Fig. 2). Only in one animal (#329) that received 50 mg of GnRH, ovulation and the emergence of a new follicular wave occurred 72 h after GnRH injection. In the other two (#343 and #334) in this group, the follicle persisted until norgestomet withdrawal. In this group, ovulation occurred at 72 h, in two females (#329 and #343), and 120 h, in one female (#334), after norgestome removal. The diameter of the ovulatory follicle in 50, 100 mg-GnRH and control groups was 13:3 1:45 mm, 12:3 0:85 mm and 12:7 0:85 mm, respectively (P > 0:05). Conclusively, the average diameter of the ovulatory dominant follicle in river buffalo was 12:7 0:54 mm in this study.
4. Discussion This study was done in order to determine the response of follicles to different doses of GnRH. In order to reduce the variability in follicle response to GnRH, a persistent dominant follicle was induced after replacement of corpus luteum with norgestomet implant, as previously used in cattle [25]. Following such treatment, all of the largest ovarian follicles, present at the time of norgestomet insertion, were maintained and displayed progressive growth and attained the diameter of 11:9 0:47 mm at the time of GnRH injection (Day 6 after norgestomet insertion). The ovarian dominant follicle of those in the control group persisted and became an ovulatory dominant follicle after norgestomet implant withdrawal. This confirms the previous findings in cattle in which the replacement of corpus luteum with the low plasma
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concentration of progestogen increased the pulse frequency of LH followed by persistence of the dominant follicle [25,33,34,35,40]. GnRH administration in cattle [9,10,32] and buffalo [1,12] resulted in the release of large amounts of FSH and LH within 4 h after treatment. The release of LH following GnRH injection either induces the ovulation of the growing dominant follicle occurring about 48 h after GnRH administration [30,37], or luteinizes and causes regression of the follicle [21] in cattle. It is also demonstrated that GnRH may induce the emergence of a new follicular wave [45] by the release of FSH, either directly [9,32] or indirectly, through the removal of the inhibitory effect of ovarian dominant follicle secreting estradiol and inhibin [7,9,16,20]. The time of ovulation and emergence of a new follicular wave, following GnRH injection in river buffalo in this study, is similar to that of the previous findings in cattle. Ovulation occurred only in one female that received 50 mg GnRH (n ¼ 3), whereas all of the females ovulated following an injection of 100 mg GnRH (n ¼ 4). In all cases ovulation occurred within 48 h and a new follicular wave emerged 72 h after GnRH injection. The response to GnRH is dependent on the stage of follicle growth in cattle [31,32,45]. GnRH administered at the growth, static and regressing phases of the dominant follicle induced ovulation in 100, 33 and 1% of cows, respectively [38]. In another study, GnRH administration in Holstein heifers, on Days 3, 6 and 9 of the estrous cycle, was associated with ovulation in 89, 56 and 22%, respectively [22]. We have also demonstrated that, ovulation occurred only in 50% of dominant follicles when GnRH is injected during the growth phase in Holstein heifers [23]. It was suggested that the ovarian response to GnRH in cows is better than that of heifers [31]. The lower response of the follicle at the static and regressing phases to GnRH was attributed to the low number of LH receptors in these follicles [16,32]. In the present study, the induction of a persistent dominant follicle reduced the variability in ovarian follicle response to GnRH, as such all buffaloes that received 100 mg GnRH ovulated. The lower number of ovulations in the females that received 50 mg GnRH, revealed that 100 mg of Gonadorelin (Fertagyl) is the effective dose of GnRH for inducing ovulation in river buffalo. The diameter of the ovarian follicle at the time of GnRH injection was 11:9 0:47 mm in this study. The ovulation of this follicle in response to 100 mg GnRH indicated that the size, as well as the stage of the ovarian follicle was appropriate at the time of GnRH injection. Baruselli et al. [5]and Berber et al. [8] found that the size of the ovarian follicle, in river buffalo, that ovulated following GnRH injection was 9:5 1:7 mm and 9:2 1:7 mm in diameter, respectively. The comparison between different doses of GnRH in ovsynch programs without considering the ovarian follicle status at the time of treatment was investigated in cattle. According to Fricke et al. [13], there was no difference in estrus synchronization rate and fertility following the use of 50 and 100 mg of cystorelin. Moreover, Yamada et al. [46] found a similar pattern of LH surge and fertility following injections of 50 and 100 mg fertirelin. To the best of our knowledge, there is no information on the effect of different doses of GnRH in buffalo. According to the result of the present study, 100 mg of Gonadorelin (Fertagyl) is the effective dose for inducing ovulation of the growing dominant follicle in river buffalo. Based on the molecular structure, analogues of GnRH have different biological half-life and potential activity. Buserelin is the most potent GnRH analogue compared to Fertirelin and Gonadorelin. Fifty micrograms Fertirelin is equivalent to 10 mg Buserelin and 500 mg
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Gonadorelin [9,26]. Therefore, more studies are needed to find out the minimum effective dose of the other GnRH analogues in this species. In this study, an injection of 100 mg Gonadorelin resulted in the ovulation of the dominant follicle followed by the formation of corpus luteum. A significant rise in progesterone concentration was detected on Day 3 followed by the formation of corpus luteum (12:59 0:29 mm in diameter) secreting 1 0:07 ng/ml of progesterone on Day 6 after GnRH injection. This corpus luteum regressed and plasma progesterone concentration dropped significantly after prostaglandin injection. The increase in plasma progesterone concentration on Day 3 after GnRH injection and the formation of the induced corpus luteum have been reported in cattle [44], which confirms the results of this study. Although the diameter of the induced corpus luteum was smaller than the natural one in cattle [31]. The diameter of the dominant follicle prior to ovulation was 12:7 0:54 mm in this study. This is similar to the previous report in this species (Berber et al. [8]; 12:9 0:9 mm). Baruselli et al. [6] found that the diameter of the ovulatory follicle in buffaloes with two or three follicular waves was 15:5 0:6 mm and 13:4 0:13 mm, respectively. Similar result was found by Taneja et al. [42] in this species (15:3 0:3 mm). In conclusion, ovarian follicle in river buffalo was maintained following the replacement of corpus luteum with norgestomet implant. Consequently, a dominant follicle ovulated in response to 100 mg Gonadorelin (within 48 h) followed by the emergence of a new follicular wave (within 72 h) after GnRH injection.
Acknowledgements Research was funded by Animal Sciences Research Institute, the Research Deputy of the University of Tehran (Project No: 218/4/612) and the Center of Excellence for Veterinary Clinical Sciences, Iranian Ministry of Science, Research and Technology. The authors would like to thank the director and station staff of Buffalo Breeding and Extension Training Center for providing facilities and kind assistance throughout the experiment and The Intervet Company, Holland, for kind provision of pharmaceutical materials to perform this experiment.
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The effect of different doses of Gonadorelin on ovarian follicle dynamics in river buffalo (Bubalus bubalis) A. Rastegarniaa,1, A. Niasari-Naslajia,b,*, P. Hovareshtia, F. Sarhaddib, M. Safaeib a
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, P.O. Box 14155-6453, Tehran, I.R. Iran b Animal Sciences Research Institute, Karaj, I.R. Iran
Received 25 September 2003; received in revised form 18 November 2003; accepted 17 January 2004
Abstract The objective of this study was to determine the response of the ovarian dominant follicle to the different doses of GnRH in river buffalo. The estrous cycle of 12 river bufflaloes was synchronized using norgestomet implant for 12 days in association with two injections of prostaglandin F2a analogue on Days 0 and 7 of implant insertion. On Day 6 or 7 of the ensuing cycle (Day 0 of the experiment), females received a norgestomet implant in conjunction with two prostaglandin injections on Days 0 and 1. On Day 6 of the experiment, females were randomly allocated into three groups. At this time, Group 1 and 2 females were given an i.m. injection of 50 or 100 mg Gonadorelin, respectively. Group 3 females did not receive any further treatment and were considered as control. All females were given prostaglandin on Day 12 and implants were removed on Day 13 of the experiment. The results revealed that in the control group, ovarian dominant follicle became persistent throughout the experiment; whereas, the persistent dominant follicle in all females belonging to Group 2 (100 mg GnRH) and one female in Group 1 (50 mg GnRH) ovulated within 48 h, subsequent with an emergence of a new follicular wave and an increase in plasma progesterone concentration within 72 and 96 h after GnRH injection, respectively. In conclusion, 100 mg of Gonadorelin seems to be the most effective dose to induce ovulation followed by an emergence of a new follicular wave in river buffalo. # 2004 Elsevier Inc. All rights reserved. Keywords: River buffalo; GnRH; Effective dose; Dominant follicle
* Corresponding author. Tel.: þ98-21-6923510; fax: þ98-21-6933222. E-mail address: [email protected] (A. Niasari-Naslaji). 1 Present address: Azad Islamic University, Urmia, West Azerbaijan, Iran.
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.01.014
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1. Introduction GnRH and GnRH analogues have been used in river buffaloes to reduce post-partum anestrus period [2,3,17,24,28,39,41], induce ovulation at the end of superovulation programs [18,27,43] and synchronize follicular emergence and ovulation in estrus synchronization programs and fixed time insemination protocols [4,8]. Different doses of GnRH have been used in these studies; however, there is no published controlled study describing the effects of different doses of GnRH on ovarian follicle growth in cyclic river buffalo. The use of the minimum effective dose of GnRH will enhance the cost-effectiveness of reproductive programs in the buffalo industry. The objective of the present study was to determine the minimum effective dose of GnRH to control the ovarian follicle growth in this species.
2. Materials and methods 2.1. Experimental location The experiment was conducted at the Buffalo Breeding and Extension Training Center, Urmia, West Azerbaijan, Iran (Latitude: 380 2300 N; Longitude: 470 4000 E; Altitude: 1568.5 m) during March–May 2002. 2.2. Experimental animals Twelve cyclic buffaloes, 10:4 1:36 years of age, weighing 498 33:04 kg LW, 224:2 48:66 days post-partum and with regular estrous cycles were selected for this study. They received a ration of alfalfa hay and concentrate including 50% barley, 15% cotton seed meal, 33% bran, 1% salt and 1% DCP. 2.3. Experimental design The estrous cycles of females were synchronized using a norgestomet implant (3 mg,17a-acetoxy-11b-methyl-19-norpreg-4-en-3,20-dione, Crestar1, Intervet, Holland) for 12 days in association with two injections of a synthetic prostaglandin F2a analogue (PGF2a, Luprostiol, Prosolvin1, Intervet, Holland) on Days 0 and 7 of the norgestomet insertion. On Days 6–7 of the ensuing cycle (Day 0 of the experiment), each received the norgestomet implant and two consecutive injections of PGF2a on Days 0 and 1 of the experiment. On Day 6 of the experiment, they were randomly assigned into three equal groups. Those in Group 1 (n ¼ 4) and Group 2 (n ¼ 4) received 50 or 100 mg GnRH analogue, i.m. (Gonadorelin, Fertagyl1, Intervet, Holland), respectively. Those in Group 3 (n ¼ 4) were not treated with GnRH and were considered as the control group. All cows were treated with PGF2a analogue on Day 12, and norgestomet implant was removed on Day 13 of the experiment.
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2.4. Ultrasound examination Ultrasound examination of the ovarian follicles was conducted on a daily basis starting from Day 0 of the experiment to the day of ovulation. Ovarian ultrasonography was performed as previously described [29] using a real time, B-mode scanner (Pie-Medical 480, Holland) equipped with a 5 MHz linear-array rectal transducer. During each examination, drawings of the ovaries were made, recording the location and the diameter of the individually identified follicles, 4 mm in diameter [19] and corpus luteum. A follicle, 10 mm in diameter, was considered morphologically to be a dominant follicle. The day of the emergence of a new follicular wave was defined as the day that the dominant follicle was, retrospectively, identified at a diameter of 4–5 mm [15,19]. The time that the dominant follicle disappeared was considered as the day of ovulation. 2.5. Blood sampling and plasma progesterone assay Daily blood sampling for the determination of plasma progesterone concentrations was performed from Day 0 of the experiment to the day of ovulation. Blood samples were collected by jugular venipuncture into 10 ml vacutainer heparinized tubes (Pars Khavar, Iran). Collected blood samples were centrifuged immediately at 2500 rpm for 15 min and plasma samples were stored at 20 8C until hormone analyses. Plasma concentrations of progesterone were determined using an extraction, single antibody radioimmunoassay [11]. The sensitivity of assay was 0.2 ng/ml and the inter- and intra-assay coefficients of variation were 10.2% (n ¼ 8) and 8.3% (n ¼ 8), respectively. 2.6. Statistical analyses Changes in the plasma concentrations of progesterone and the diameter of ovarian follicles over time were analyzed for the effects of treatments, day and treatment by day interaction using procedure GLM in SAS/STAT [36] by either univariate or multivariate analysis [14], with repeated measures analysis included in the model. Multivariate analyses were used where variance and covariance structures over time did not conform to the analysis of variance assumptions, using sphericity test. Single-points measurements for follicular characteristics and wave emergence were compared using ANOVA followed by Tukey’s Honestly significant difference [47]. In case the assumptions of parametric tests were not achieved, the non-parametric ANOVA for a single factor (Kruskal–Wallis one-way ANOVA) of SAS/STAT was used. Data were presented as mean S:E:M:
3. Results As all experimental groups received the same treatment until Day 6 of the experiment (the day of GnRH injection), all data for this period of experiment were pooled and analyzed together.
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3.1. Plasma progesterone concentration The concentration of plasma progesterone was monitored from Day 0 (Days 6–7 of estrous cycle ¼ day of norgestomet insertion) to Day 16 of the experiment (Fig. 1). This was 1:9 0:28 ng/ml at the time of norgestomet insertion, which was reduced to a value below the sensitivity of the progesterone assay within 24 h after the first prostaglandin injection and remained unchanged until Day 6 of the experiment. In the control group females, that did not receive further treatment, the plasma progesterone concentrations remained constant at the basal concentration throughout the experiment. Following GnRH administration, in only one animal (#329) which was in the group that received 50 mg Gonadorelin, the plasma concentration of progesterone increased significantly (P < 0:05) at 96 h after GnRH injection and this trend continued until Day 12 of the experiment (day of prostaglandin injection). In all females receiving 100 mg Gonadorelin, the plasma progesterone concentration started to increase at 96 h after treatment (0:53 0:06 ng/ml; P < 0:05) and reached a concentration of 1 0:07 ng/ml (P < 0:05) at the time of PGF2a treatment (Day 12 of the experiment). It decreased to a value below the sensitivity of assay, 24 h after prostaglandin injection (Fig. 1). 3.2. Ovarian follicle dynamics On Day 0 of the experiment (Days 6–7 of the estrous cycle), 10 females had an ovarian follicle 10 mm (10:9 0:36 mm in diameter), and all of them had a corpus luteum (17:4 1:38 mm), which regressed following two injections of PGF2a on Days 0 and 1. From Days 0 to 6 (day of GnRH injection) of the experiment, the ovarian follicles progressively grew in all animals (P < 0:05; Fig. 2). At the time of GnRH treatment (Day 6), 11 cows had a dominant follicle (11:9 0:47 mm). Only one, in the group that received 50 mg of GnRH, did not have dominant follicle; therefore, it was excluded from the data set. In all animals in the control group, the ovarian follicle maintained and became ovulatory. In
Fig. 1. The plasma progesterone concentrations from Day 0 (day of norgestomet insertion) to Day 15 of the experiment in river buffaloes that received 0, 50 and 100 mg GnRH on Day 6 of the experiment.
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Fig. 2. The growth pattern of ovarian follicle from Day 0 (day of norgestomet insertion) to Day 16 of the experiment in river buffaloes that received 0, 50 and 100 mg GnRH on Day 6 of the experiment.
contrast, the dominant follicle in the group that received 100 mg GnRH ovulated and corpus luteum was detected at 72 h after GnRH injection. The diameter of the induced corpus luteum was 12:5 0:29 mm at the time of PGF2a treatment (Day 6 after GnRH injection). In this group, the emergence of a new follicular wave occurred at 72 h after GnRH injection. Ovulation of the dominant follicle in control and 100 mg GnRH groups was detected between 72 and 96 h (three females at 72 h and one female at 96 h) after norgestomet withdrawal (Fig. 2). Only in one animal (#329) that received 50 mg of GnRH, ovulation and the emergence of a new follicular wave occurred 72 h after GnRH injection. In the other two (#343 and #334) in this group, the follicle persisted until norgestomet withdrawal. In this group, ovulation occurred at 72 h, in two females (#329 and #343), and 120 h, in one female (#334), after norgestome removal. The diameter of the ovulatory follicle in 50, 100 mg-GnRH and control groups was 13:3 1:45 mm, 12:3 0:85 mm and 12:7 0:85 mm, respectively (P > 0:05). Conclusively, the average diameter of the ovulatory dominant follicle in river buffalo was 12:7 0:54 mm in this study.
4. Discussion This study was done in order to determine the response of follicles to different doses of GnRH. In order to reduce the variability in follicle response to GnRH, a persistent dominant follicle was induced after replacement of corpus luteum with norgestomet implant, as previously used in cattle [25]. Following such treatment, all of the largest ovarian follicles, present at the time of norgestomet insertion, were maintained and displayed progressive growth and attained the diameter of 11:9 0:47 mm at the time of GnRH injection (Day 6 after norgestomet insertion). The ovarian dominant follicle of those in the control group persisted and became an ovulatory dominant follicle after norgestomet implant withdrawal. This confirms the previous findings in cattle in which the replacement of corpus luteum with the low plasma
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concentration of progestogen increased the pulse frequency of LH followed by persistence of the dominant follicle [25,33,34,35,40]. GnRH administration in cattle [9,10,32] and buffalo [1,12] resulted in the release of large amounts of FSH and LH within 4 h after treatment. The release of LH following GnRH injection either induces the ovulation of the growing dominant follicle occurring about 48 h after GnRH administration [30,37], or luteinizes and causes regression of the follicle [21] in cattle. It is also demonstrated that GnRH may induce the emergence of a new follicular wave [45] by the release of FSH, either directly [9,32] or indirectly, through the removal of the inhibitory effect of ovarian dominant follicle secreting estradiol and inhibin [7,9,16,20]. The time of ovulation and emergence of a new follicular wave, following GnRH injection in river buffalo in this study, is similar to that of the previous findings in cattle. Ovulation occurred only in one female that received 50 mg GnRH (n ¼ 3), whereas all of the females ovulated following an injection of 100 mg GnRH (n ¼ 4). In all cases ovulation occurred within 48 h and a new follicular wave emerged 72 h after GnRH injection. The response to GnRH is dependent on the stage of follicle growth in cattle [31,32,45]. GnRH administered at the growth, static and regressing phases of the dominant follicle induced ovulation in 100, 33 and 1% of cows, respectively [38]. In another study, GnRH administration in Holstein heifers, on Days 3, 6 and 9 of the estrous cycle, was associated with ovulation in 89, 56 and 22%, respectively [22]. We have also demonstrated that, ovulation occurred only in 50% of dominant follicles when GnRH is injected during the growth phase in Holstein heifers [23]. It was suggested that the ovarian response to GnRH in cows is better than that of heifers [31]. The lower response of the follicle at the static and regressing phases to GnRH was attributed to the low number of LH receptors in these follicles [16,32]. In the present study, the induction of a persistent dominant follicle reduced the variability in ovarian follicle response to GnRH, as such all buffaloes that received 100 mg GnRH ovulated. The lower number of ovulations in the females that received 50 mg GnRH, revealed that 100 mg of Gonadorelin (Fertagyl) is the effective dose of GnRH for inducing ovulation in river buffalo. The diameter of the ovarian follicle at the time of GnRH injection was 11:9 0:47 mm in this study. The ovulation of this follicle in response to 100 mg GnRH indicated that the size, as well as the stage of the ovarian follicle was appropriate at the time of GnRH injection. Baruselli et al. [5]and Berber et al. [8] found that the size of the ovarian follicle, in river buffalo, that ovulated following GnRH injection was 9:5 1:7 mm and 9:2 1:7 mm in diameter, respectively. The comparison between different doses of GnRH in ovsynch programs without considering the ovarian follicle status at the time of treatment was investigated in cattle. According to Fricke et al. [13], there was no difference in estrus synchronization rate and fertility following the use of 50 and 100 mg of cystorelin. Moreover, Yamada et al. [46] found a similar pattern of LH surge and fertility following injections of 50 and 100 mg fertirelin. To the best of our knowledge, there is no information on the effect of different doses of GnRH in buffalo. According to the result of the present study, 100 mg of Gonadorelin (Fertagyl) is the effective dose for inducing ovulation of the growing dominant follicle in river buffalo. Based on the molecular structure, analogues of GnRH have different biological half-life and potential activity. Buserelin is the most potent GnRH analogue compared to Fertirelin and Gonadorelin. Fifty micrograms Fertirelin is equivalent to 10 mg Buserelin and 500 mg
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Gonadorelin [9,26]. Therefore, more studies are needed to find out the minimum effective dose of the other GnRH analogues in this species. In this study, an injection of 100 mg Gonadorelin resulted in the ovulation of the dominant follicle followed by the formation of corpus luteum. A significant rise in progesterone concentration was detected on Day 3 followed by the formation of corpus luteum (12:59 0:29 mm in diameter) secreting 1 0:07 ng/ml of progesterone on Day 6 after GnRH injection. This corpus luteum regressed and plasma progesterone concentration dropped significantly after prostaglandin injection. The increase in plasma progesterone concentration on Day 3 after GnRH injection and the formation of the induced corpus luteum have been reported in cattle [44], which confirms the results of this study. Although the diameter of the induced corpus luteum was smaller than the natural one in cattle [31]. The diameter of the dominant follicle prior to ovulation was 12:7 0:54 mm in this study. This is similar to the previous report in this species (Berber et al. [8]; 12:9 0:9 mm). Baruselli et al. [6] found that the diameter of the ovulatory follicle in buffaloes with two or three follicular waves was 15:5 0:6 mm and 13:4 0:13 mm, respectively. Similar result was found by Taneja et al. [42] in this species (15:3 0:3 mm). In conclusion, ovarian follicle in river buffalo was maintained following the replacement of corpus luteum with norgestomet implant. Consequently, a dominant follicle ovulated in response to 100 mg Gonadorelin (within 48 h) followed by the emergence of a new follicular wave (within 72 h) after GnRH injection.
Acknowledgements Research was funded by Animal Sciences Research Institute, the Research Deputy of the University of Tehran (Project No: 218/4/612) and the Center of Excellence for Veterinary Clinical Sciences, Iranian Ministry of Science, Research and Technology. The authors would like to thank the director and station staff of Buffalo Breeding and Extension Training Center for providing facilities and kind assistance throughout the experiment and The Intervet Company, Holland, for kind provision of pharmaceutical materials to perform this experiment.
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