Theriogenology 83 (2015) 192–198
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Effect of GnRH treatment on ovarian activity and reproductive performance of low-prolific Rahmani ewes N.M. Hashem*, K.M. El-Azrak, A.N.M. Nour El-Din, T.A. Taha, M.H. Salem Animal Production Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 7 September 2014 Accepted 10 September 2014
This study was designed to evaluate the effect of GnRH treatment during different times of the reproductive cycle on ovarian activity, progesterone (P4) concentration, and subsequent fertility of low-prolific, subtropical, Rahmani ewes during breeding season. Fortyfive ewes were synchronized for estrus using a double injection of 0.5 mL of PGF2a agonist (125-mg cloprostenol), 11 days apart. Ewes showing estrus (Day 0) were treated with 1 mL of GnRH agonist (4-mg buserelin) on the day of estrus (GnRH0, n ¼ 12) or 7 days post-mating (GnRH7, n ¼ 10) or on both days (GnRH0þ7, n ¼ 11) or not (control, n ¼ 12). Ovarian response to the treatment and diagnosis of pregnancy were ultrasonographically monitored. Also, serum P4 concentration was determined weekly throughout 28 days postmating. Results showed that neither total number of follicles nor their populations were changed on Day 0 or 7 days post-mating by the GnRH treatment. GnRH treatment on Day 0 or Day 7 post-mating or both days did not enhance ovulation rate compared with the control. The mean numbers of accessory CL increased (P < 0.05) in the GnRH7 group than those in the control and GnRH0 groups, whereas it was intermediate in the GnRH0þ7 group. The greatest (P < 0.05) overall mean of serum P4 concentration was for the GnRH7 and GnRH0þ7 groups, followed by the GnRH0 and control groups. Serum P4 concentration increased (P < 0.05) on Day 14 post-mating and continued higher (P < 0.05) until Day 28 post-mating in the GnRH7 and GnRH0þ7 groups compared with the control. Regardless of the time of GnRH administration, GnRH treatment reduced (P < 0.05) pregnancy loss from Day 40 post-mating to parturition and tended to enhance (P < 0.20) lambing rate compared with the control. In conclusion, a single dose of GnRH at the time of estrus or 7 days post-mating could be used as an effective protocol to decrease pregnancy loss from Day 40 after mating to parturition in low-prolific Rahmani ewes. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ovulation rate GnRH Embryonic mortality Progesterone Rahmani ewe
1. Introduction In sheep breeds kept for meat production, litter size or lamb crop is an important economic trait. Sheep breeds raised in Egypt are mainly used for meat production. However, they are classified as low-prolific breeds [1]. This may be due to the decrease in follicular population [2] and thus low ovulation rate [3,4] or occurrence of early
* Corresponding author. Tel.: þ20 35921960; fax: þ20 35922780. E-mail address: [email protected] (N.M. Hashem). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.016
embryonic and/or fetal losses, as in different sheep breeds, fertilization failure accounts for only 5% to 10% of losses [5]. Most of the losses are the result of embryonic mortality [6]. In sheep, 30% to 40% of fertilized ova are lost during the preimplantation period and 70% to 80% of losses are during the first 3 weeks post-mating [7]. Accordingly, increasing ovulation rate or decreasing embryonic losses are the major important traits that should be improved for enhancing prolificacy of ewes. Treatment with GnRH agonists results in an endogenous release of LH surge within 1 to 4 hours post-administration [8,9]. This surge is pivotal for the occurrence of many critical reproductive events related to
N.M. Hashem et al. / Theriogenology 83 (2015) 192–198
subsequent fertility such as the development of dominant follicle, oocyte maturation, ovulation, and luteinization [10,11]. Exogenous GnRH treatment immediately after insemination increased the multiple birth rates of lowprolific Awassi ewes by enhancing ovulation [12]. Also, GnRH administration at the time of mating [13] or before maternal recognition of pregnancy [14] improves luteal functions and induces luteinization of developing follicles, which increases circulating progesterone levels and thereby embryo survival in many ruminant species [11,13,15].The present study was designed to examine the effect of GnRH administration at different times of the reproductive cycle including the day of estrus or 7 days post-mating or both days on reproductive performance of low-prolific Rahmani ewes.
193
(control, n ¼ 12) and received an im injection of 1 mL of saline (0.9% NaCl, placebo). The other three groups were classified according to the time of GnRH treatment. An im injection of 1 mL of GnRH agonist (4-mg buserelin/mL; Receptal, Boxmeer, Holland) was applied on the day of estrus (Day 0, GnRH0 group, n ¼ 12), 7 days post-mating (Day 7, GnRH7 group, n ¼ 10) or on both days (0 and 7, GnRH0þ7 group, n ¼ 11). Gonadotropin-releasing hormone treatment on the day of estrus (Day 0) was applied once the signs of estrus were detected, and the ultrasonography examination was performed. Then, ewes were subjected to an assisted natural mating by proven-fertile rams (1 ram/ 10–12 ewes). Each ram was allowed to mate equal numbers of ewes from each group to eliminate the ram effect. 2.3. Ultrasonography examination
2. Materials and methods This study was conducted at the Agricultural Experimental Station (31 200 N, 30 E), Faculty of Agriculture, Alexandria University. The experiment was carried out during the period from August (estrus synchronization and mating) to January (lambing) representing the breeding season, whereas this breed tends to breed all the year round with a peak of estrus activity throughout summer and autumn, associating with long photoperiod, high temperature, and high relative humidity [2]. All procedures and experimental protocols were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching, Federation of Animal Science Societies [16]. 2.1. Animals and management Forty-five multiparous, fertile, nonlactating Rahmani ewes aging 2.9 0.25 years, weighing 43.46 0.78 kg, and with body condition scores of 2.66 0.17 (scale ranging from 1 ¼ emaciated to 5 ¼ obese; [17]) at allocation were used in the present study. All ewes were kept outdoors with a shelter during the day and were housed in a semiopen barn at the night. Ewes were fed according to their body weight requirements [18]. Chopped green maize in addition to hay was offered in summer and autumn and Egyptian clover (Trifolium alexandrinum) in winter. Each animal also received 750 g/day of a pelleted concentrate mixture that contained 68% total digestible nutrients and 14% crude protein. Water was offered to animals as a free choice. All ewes were clinically normal with a healthy appearance. 2.2. Experimental design All animals were synchronized for estrus using a double intramuscular (im) injection of 0.5-mL PGF2a (Estrumate, 250-mg cloprostenol/mL, Schering-Plough Animal Health, Germany), 11 days apart. After 24 hours of the end of the estrus synchronization protocol (second PGF2a injection), all ewes were observed twice daily (at 7 AM and 2 PM) for overt signs of estrus using teaser rams for 5 consecutive days. Ewes displaying behavioral estrus were divided into four homogenous experimental groups, with respect to body weight and parity. The first group served as control
An ultrasound, real-time, B-mode scanner equipped with 5- and 7.5-MHz, linear-array, endorectal probes (Pie Medical Equipment B.V., Maastricht, Netherlands) was used to determine the ovarian response to GnRH treatment. The scanning procedure described by Gonzalez-Bulnes et al. [19] was used. Briefly, animals were banned from feed for 12 hours before examination, which was carried out in a standing position. The probe was fitted to a plastic rod (1 30 cm) as an adapter to enable the insertion of the probe into the rectum. The probe was lubricated by a hydrosoluble gel and sheathed with polyvinyl chloride pipe (2 35 cm) to avoid damage of the rectal mucosa and was gently inserted about 20 cm through the rectum after feces removal until the anechoic content of the bladder was visible on the screen, and then the probe was rotated 90 clockwise and 180 counterclockwise across the reproductive tract until the uterine horns and both ovaries were scanned. Ovarian activity including total number of follicles 2 mm or greater/ewe and follicle population (i.e., small follicles, 2 to 3 mm; medium follicles, > 3 to < 5 mm; and large [ovulatory] follicles, >5 mm) [3,4] in each ewe was measured and recorded twice on Days 0 and 7 (before GnRH administration). The diameter of the largest (ovulatory) follicle was also measured on Day 0 (day of estrus). Also, numbers and diameters of CLs were recorded on Days 7 and 14 post-mating. The CLs (main CL) observed on Day 7 post-mating (formed due to ovulation of LH-responsive follicles raised on the day of estrus) were expressed as ovulation rate (number of CLs detected on Day 7/total number of ewes in each group), whereas those observed for the first time on Day 14 post-mating were considered as accessory CLs (number of CLs detected on Day 14/total number of ewes in each group). Pregnancy diagnosis and number of embryos per each ewe were recorded by scanning the uterine contents at 28 and 40 days post-mating. 2.4. Blood collection and hormonal assessment Blood samples were collected into 10-mL nonheparinized tubes by means of jugular venipuncture on Days 7, 14, 21, and 28 post-mating. All samples were collected in the morning before access to feed and water. Serum was obtained by centrifugation of samples at 700g for 20 minutes and was stored at 20 C until analysis. Blood serum
194
N.M. Hashem et al. / Theriogenology 83 (2015) 192–198
concentration of progesterone (P4) was measured using solid-phase enzyme immunoassay kits obtained from Monobind Inc., USA. The lower limit of detection (95% binding/0% binding [B/B0]) was 0.10 ng/mL serum and the intra-assay and interassay coefficients of variation were 5.1% and 7.5%, respectively. 2.5. Assessment of reproductive performance The reproductive performance of ewes was evaluated by calculating the following variables: estrus rate ¼ (number of ewes displaying estrus/number of synchronized ewes) 100; conception rate ¼ (number of ewes conceiving on Day 28 or 40/number of exposed ewes) 100; lambing rate ¼ (number of ewes lambing/number of exposed ewes) 100; pregnancy loss before Day 40 (40 days) postmating ¼ (number of ewes nonlambing/number of ewes conceiving on Day 40) 100; embryonic mortality ¼ [(number of detected embryos on Day 28 number of detected embryos on Day 40)/number of detected embryos on Day 28] 100; fetal mortality ¼ [(number of detected embryos on Day 40 number of lambs born)/number of detected embryos on Day 40] 100; and fecundity rate ¼ (number of lambs born/number of exposed ewes) 100. 2.6. Statistical analysis Total number of follicles and their classifications (according to diameter) and numbers of CL were subjected to square root transformation to approximate normal distribution [20] before subjecting to ANOVA. Data observed once a time including the diameter of dominant follicles, ovulation rate, and the number of accessory CL and their diameters were analyzed by generalized linear model procedure of SAS [21] using the following model, yij ¼ mþ Ti þ eij in which yij is the observed value of the dependent variable, m is the overall mean, Ti is the fixed effect of the ith treatment (i ¼ 1:4), and eij is the residual error. Best linear estimation was used for the estimation of fixed effects. Least squares procedures using a mixed model [21], considering the day effect as repeated measurements, was used. The GnRH treatment and the day of cycle were used as fixed effects and individual ewes as random effects. To study the effects of GnRH treatment, day of estrous cycle and their interaction on total number of follicles, follicle population, diameter of main CL, and serum progesterone
concentrations, this mixed model was used, yijk ¼ mþ Ti þ Dj þ (TD)ij þ eijk in which yijk is the observed value of the dependent variable determined from a sample taken from each animal, m is the overall mean, Ti is the fixed effect of the ith treatment (i ¼ 1:4), Dj is the fixed effect of the jth day of estrous cycle (j ¼ 1:2 for follicle parameters and diameter of main CL and 1:4 for serum progesterone concentration), (TD)ij is the interaction between treatment and day of estrous cycle, and eijk is the residual error. Differences among the treatment groups were detected using the Duncan new multiple range test. Categorical data were analyzed using chi-square test. All the results were expressed as the mean standard error of the mean). The statistical significance was accepted at P < 0.05.
3. Results 3.1. Effect of GnRH treatment on ovarian activity The effects of GnRH treatment, time, and treatment by time interaction on follicular characteristics are presented in Table 1. GnRH treatment and treatment by time interaction did not affect the mean of total number of follicles and follicles population. However, these traits were affected (P < 0.05) by the time of the estrous cycle. Diameter of the largest follicle recorded at the day of estrus was not affected by GnRH treatment. Data shown in Table 2 revealed that GnRH administration on Days 0 or 7 post-mating or on both days did not enhance ovulation rate compared with the control. The diameters of CL (formed due to ovulation of mature follicles of ovulatory wave) were not affected by the treatment. The mean numbers of accessory CL increased (P < 0.05) in the GnRH7 group followed by the GnRH0þ7 group, whereas none of such structure was observed in the control and GnRH0 groups. Diameters of accessory CL did not differ (P > 0.05) between the two treated groups in which they were observed. Serum P4 concentration was affected (P < 0.05) by the GnRH treatment (Table 2) and interaction of treatment by time (Fig. 1). The greatest (P < 0.05) values of P4 concentration were recorded for the GnRH7 and GnRH0þ7 groups compared with those of GnRH0 group; however, both of them were higher (P < 0.05) than that of the control group. Changes of serum P4 concentrations throughout 28 days post-mating (Fig. 1) indicated that both of the GnRH7 and GnRH0þ7 groups recorded greater (P < 0.05) values throughout 14 to 28 days post-mating
Table 1 Characterization of ovarian follicles of ewes treated with GnRH on the day of estrus (GnRH0), 7 days post-mating (GnRH7), and on both days (GnRH0þ7) compared with the control (mean standard error of the mean [SEM]). Parameter
Total number of follicles Follicle population Small, 2 to 3 mm Medium, >3 to 5 mm Diameter of the largest (ovulatory) follicle, mma a
Treatment (T)
SEM
P value T
Day (D)
TD
1.73
0.28
0.33
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Effect of GnRH treatment on ovarian activity and reproductive performance of low-prolific Rahmani ewes N.M. Hashem*, K.M. El-Azrak, A.N.M. Nour El-Din, T.A. Taha, M.H. Salem Animal Production Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 7 September 2014 Accepted 10 September 2014
This study was designed to evaluate the effect of GnRH treatment during different times of the reproductive cycle on ovarian activity, progesterone (P4) concentration, and subsequent fertility of low-prolific, subtropical, Rahmani ewes during breeding season. Fortyfive ewes were synchronized for estrus using a double injection of 0.5 mL of PGF2a agonist (125-mg cloprostenol), 11 days apart. Ewes showing estrus (Day 0) were treated with 1 mL of GnRH agonist (4-mg buserelin) on the day of estrus (GnRH0, n ¼ 12) or 7 days post-mating (GnRH7, n ¼ 10) or on both days (GnRH0þ7, n ¼ 11) or not (control, n ¼ 12). Ovarian response to the treatment and diagnosis of pregnancy were ultrasonographically monitored. Also, serum P4 concentration was determined weekly throughout 28 days postmating. Results showed that neither total number of follicles nor their populations were changed on Day 0 or 7 days post-mating by the GnRH treatment. GnRH treatment on Day 0 or Day 7 post-mating or both days did not enhance ovulation rate compared with the control. The mean numbers of accessory CL increased (P < 0.05) in the GnRH7 group than those in the control and GnRH0 groups, whereas it was intermediate in the GnRH0þ7 group. The greatest (P < 0.05) overall mean of serum P4 concentration was for the GnRH7 and GnRH0þ7 groups, followed by the GnRH0 and control groups. Serum P4 concentration increased (P < 0.05) on Day 14 post-mating and continued higher (P < 0.05) until Day 28 post-mating in the GnRH7 and GnRH0þ7 groups compared with the control. Regardless of the time of GnRH administration, GnRH treatment reduced (P < 0.05) pregnancy loss from Day 40 post-mating to parturition and tended to enhance (P < 0.20) lambing rate compared with the control. In conclusion, a single dose of GnRH at the time of estrus or 7 days post-mating could be used as an effective protocol to decrease pregnancy loss from Day 40 after mating to parturition in low-prolific Rahmani ewes. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ovulation rate GnRH Embryonic mortality Progesterone Rahmani ewe
1. Introduction In sheep breeds kept for meat production, litter size or lamb crop is an important economic trait. Sheep breeds raised in Egypt are mainly used for meat production. However, they are classified as low-prolific breeds [1]. This may be due to the decrease in follicular population [2] and thus low ovulation rate [3,4] or occurrence of early
* Corresponding author. Tel.: þ20 35921960; fax: þ20 35922780. E-mail address: [email protected] (N.M. Hashem). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.016
embryonic and/or fetal losses, as in different sheep breeds, fertilization failure accounts for only 5% to 10% of losses [5]. Most of the losses are the result of embryonic mortality [6]. In sheep, 30% to 40% of fertilized ova are lost during the preimplantation period and 70% to 80% of losses are during the first 3 weeks post-mating [7]. Accordingly, increasing ovulation rate or decreasing embryonic losses are the major important traits that should be improved for enhancing prolificacy of ewes. Treatment with GnRH agonists results in an endogenous release of LH surge within 1 to 4 hours post-administration [8,9]. This surge is pivotal for the occurrence of many critical reproductive events related to
N.M. Hashem et al. / Theriogenology 83 (2015) 192–198
subsequent fertility such as the development of dominant follicle, oocyte maturation, ovulation, and luteinization [10,11]. Exogenous GnRH treatment immediately after insemination increased the multiple birth rates of lowprolific Awassi ewes by enhancing ovulation [12]. Also, GnRH administration at the time of mating [13] or before maternal recognition of pregnancy [14] improves luteal functions and induces luteinization of developing follicles, which increases circulating progesterone levels and thereby embryo survival in many ruminant species [11,13,15].The present study was designed to examine the effect of GnRH administration at different times of the reproductive cycle including the day of estrus or 7 days post-mating or both days on reproductive performance of low-prolific Rahmani ewes.
193
(control, n ¼ 12) and received an im injection of 1 mL of saline (0.9% NaCl, placebo). The other three groups were classified according to the time of GnRH treatment. An im injection of 1 mL of GnRH agonist (4-mg buserelin/mL; Receptal, Boxmeer, Holland) was applied on the day of estrus (Day 0, GnRH0 group, n ¼ 12), 7 days post-mating (Day 7, GnRH7 group, n ¼ 10) or on both days (0 and 7, GnRH0þ7 group, n ¼ 11). Gonadotropin-releasing hormone treatment on the day of estrus (Day 0) was applied once the signs of estrus were detected, and the ultrasonography examination was performed. Then, ewes were subjected to an assisted natural mating by proven-fertile rams (1 ram/ 10–12 ewes). Each ram was allowed to mate equal numbers of ewes from each group to eliminate the ram effect. 2.3. Ultrasonography examination
2. Materials and methods This study was conducted at the Agricultural Experimental Station (31 200 N, 30 E), Faculty of Agriculture, Alexandria University. The experiment was carried out during the period from August (estrus synchronization and mating) to January (lambing) representing the breeding season, whereas this breed tends to breed all the year round with a peak of estrus activity throughout summer and autumn, associating with long photoperiod, high temperature, and high relative humidity [2]. All procedures and experimental protocols were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching, Federation of Animal Science Societies [16]. 2.1. Animals and management Forty-five multiparous, fertile, nonlactating Rahmani ewes aging 2.9 0.25 years, weighing 43.46 0.78 kg, and with body condition scores of 2.66 0.17 (scale ranging from 1 ¼ emaciated to 5 ¼ obese; [17]) at allocation were used in the present study. All ewes were kept outdoors with a shelter during the day and were housed in a semiopen barn at the night. Ewes were fed according to their body weight requirements [18]. Chopped green maize in addition to hay was offered in summer and autumn and Egyptian clover (Trifolium alexandrinum) in winter. Each animal also received 750 g/day of a pelleted concentrate mixture that contained 68% total digestible nutrients and 14% crude protein. Water was offered to animals as a free choice. All ewes were clinically normal with a healthy appearance. 2.2. Experimental design All animals were synchronized for estrus using a double intramuscular (im) injection of 0.5-mL PGF2a (Estrumate, 250-mg cloprostenol/mL, Schering-Plough Animal Health, Germany), 11 days apart. After 24 hours of the end of the estrus synchronization protocol (second PGF2a injection), all ewes were observed twice daily (at 7 AM and 2 PM) for overt signs of estrus using teaser rams for 5 consecutive days. Ewes displaying behavioral estrus were divided into four homogenous experimental groups, with respect to body weight and parity. The first group served as control
An ultrasound, real-time, B-mode scanner equipped with 5- and 7.5-MHz, linear-array, endorectal probes (Pie Medical Equipment B.V., Maastricht, Netherlands) was used to determine the ovarian response to GnRH treatment. The scanning procedure described by Gonzalez-Bulnes et al. [19] was used. Briefly, animals were banned from feed for 12 hours before examination, which was carried out in a standing position. The probe was fitted to a plastic rod (1 30 cm) as an adapter to enable the insertion of the probe into the rectum. The probe was lubricated by a hydrosoluble gel and sheathed with polyvinyl chloride pipe (2 35 cm) to avoid damage of the rectal mucosa and was gently inserted about 20 cm through the rectum after feces removal until the anechoic content of the bladder was visible on the screen, and then the probe was rotated 90 clockwise and 180 counterclockwise across the reproductive tract until the uterine horns and both ovaries were scanned. Ovarian activity including total number of follicles 2 mm or greater/ewe and follicle population (i.e., small follicles, 2 to 3 mm; medium follicles, > 3 to < 5 mm; and large [ovulatory] follicles, >5 mm) [3,4] in each ewe was measured and recorded twice on Days 0 and 7 (before GnRH administration). The diameter of the largest (ovulatory) follicle was also measured on Day 0 (day of estrus). Also, numbers and diameters of CLs were recorded on Days 7 and 14 post-mating. The CLs (main CL) observed on Day 7 post-mating (formed due to ovulation of LH-responsive follicles raised on the day of estrus) were expressed as ovulation rate (number of CLs detected on Day 7/total number of ewes in each group), whereas those observed for the first time on Day 14 post-mating were considered as accessory CLs (number of CLs detected on Day 14/total number of ewes in each group). Pregnancy diagnosis and number of embryos per each ewe were recorded by scanning the uterine contents at 28 and 40 days post-mating. 2.4. Blood collection and hormonal assessment Blood samples were collected into 10-mL nonheparinized tubes by means of jugular venipuncture on Days 7, 14, 21, and 28 post-mating. All samples were collected in the morning before access to feed and water. Serum was obtained by centrifugation of samples at 700g for 20 minutes and was stored at 20 C until analysis. Blood serum
194
N.M. Hashem et al. / Theriogenology 83 (2015) 192–198
concentration of progesterone (P4) was measured using solid-phase enzyme immunoassay kits obtained from Monobind Inc., USA. The lower limit of detection (95% binding/0% binding [B/B0]) was 0.10 ng/mL serum and the intra-assay and interassay coefficients of variation were 5.1% and 7.5%, respectively. 2.5. Assessment of reproductive performance The reproductive performance of ewes was evaluated by calculating the following variables: estrus rate ¼ (number of ewes displaying estrus/number of synchronized ewes) 100; conception rate ¼ (number of ewes conceiving on Day 28 or 40/number of exposed ewes) 100; lambing rate ¼ (number of ewes lambing/number of exposed ewes) 100; pregnancy loss before Day 40 (40 days) postmating ¼ (number of ewes nonlambing/number of ewes conceiving on Day 40) 100; embryonic mortality ¼ [(number of detected embryos on Day 28 number of detected embryos on Day 40)/number of detected embryos on Day 28] 100; fetal mortality ¼ [(number of detected embryos on Day 40 number of lambs born)/number of detected embryos on Day 40] 100; and fecundity rate ¼ (number of lambs born/number of exposed ewes) 100. 2.6. Statistical analysis Total number of follicles and their classifications (according to diameter) and numbers of CL were subjected to square root transformation to approximate normal distribution [20] before subjecting to ANOVA. Data observed once a time including the diameter of dominant follicles, ovulation rate, and the number of accessory CL and their diameters were analyzed by generalized linear model procedure of SAS [21] using the following model, yij ¼ mþ Ti þ eij in which yij is the observed value of the dependent variable, m is the overall mean, Ti is the fixed effect of the ith treatment (i ¼ 1:4), and eij is the residual error. Best linear estimation was used for the estimation of fixed effects. Least squares procedures using a mixed model [21], considering the day effect as repeated measurements, was used. The GnRH treatment and the day of cycle were used as fixed effects and individual ewes as random effects. To study the effects of GnRH treatment, day of estrous cycle and their interaction on total number of follicles, follicle population, diameter of main CL, and serum progesterone
concentrations, this mixed model was used, yijk ¼ mþ Ti þ Dj þ (TD)ij þ eijk in which yijk is the observed value of the dependent variable determined from a sample taken from each animal, m is the overall mean, Ti is the fixed effect of the ith treatment (i ¼ 1:4), Dj is the fixed effect of the jth day of estrous cycle (j ¼ 1:2 for follicle parameters and diameter of main CL and 1:4 for serum progesterone concentration), (TD)ij is the interaction between treatment and day of estrous cycle, and eijk is the residual error. Differences among the treatment groups were detected using the Duncan new multiple range test. Categorical data were analyzed using chi-square test. All the results were expressed as the mean standard error of the mean). The statistical significance was accepted at P < 0.05.
3. Results 3.1. Effect of GnRH treatment on ovarian activity The effects of GnRH treatment, time, and treatment by time interaction on follicular characteristics are presented in Table 1. GnRH treatment and treatment by time interaction did not affect the mean of total number of follicles and follicles population. However, these traits were affected (P < 0.05) by the time of the estrous cycle. Diameter of the largest follicle recorded at the day of estrus was not affected by GnRH treatment. Data shown in Table 2 revealed that GnRH administration on Days 0 or 7 post-mating or on both days did not enhance ovulation rate compared with the control. The diameters of CL (formed due to ovulation of mature follicles of ovulatory wave) were not affected by the treatment. The mean numbers of accessory CL increased (P < 0.05) in the GnRH7 group followed by the GnRH0þ7 group, whereas none of such structure was observed in the control and GnRH0 groups. Diameters of accessory CL did not differ (P > 0.05) between the two treated groups in which they were observed. Serum P4 concentration was affected (P < 0.05) by the GnRH treatment (Table 2) and interaction of treatment by time (Fig. 1). The greatest (P < 0.05) values of P4 concentration were recorded for the GnRH7 and GnRH0þ7 groups compared with those of GnRH0 group; however, both of them were higher (P < 0.05) than that of the control group. Changes of serum P4 concentrations throughout 28 days post-mating (Fig. 1) indicated that both of the GnRH7 and GnRH0þ7 groups recorded greater (P < 0.05) values throughout 14 to 28 days post-mating
Table 1 Characterization of ovarian follicles of ewes treated with GnRH on the day of estrus (GnRH0), 7 days post-mating (GnRH7), and on both days (GnRH0þ7) compared with the control (mean standard error of the mean [SEM]). Parameter
Total number of follicles Follicle population Small, 2 to 3 mm Medium, >3 to 5 mm Diameter of the largest (ovulatory) follicle, mma a
Treatment (T)
SEM
P value T
Day (D)
TD
1.73
0.28
0.33
