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Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis)
Animal Reproduction Science 123 (2011) 169–174
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis) A.K. Pandey a,∗ , G.S. Dhaliwal b , S.P.S. Ghuman a , S.K. Agarwal c a
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India Department of Teaching Veterinary Clinical Complex, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India c Division of Animal Reproduction, Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India b
a r t i c l e
i n f o
Article history: Received 31 August 2010 Received in revised form 21 November 2010 Accepted 6 December 2010 Available online 14 December 2010 Keywords: Buffalo Estradiol Luteal profile Pre-ovulatory follicle Progesterone
a b s t r a c t The present study was designed to investigate the impact of pre-ovulatory follicle (POF) diameter on the day of estrus on plasma estradiol concentration, subsequent luteal profile (corpus luteum, CL, diameter and plasma progesterone concentration) and conception rate in buffaloes. Twenty-eight buffaloes were synchronized with synthetic analogue of prostaglandin F2␣ (PGF2␣ ) administered 11 days apart. Transrectal ultrasonography and jugular vein blood sampling was carried out on the day of estrus and on days 0 (day of ovulation), 5, 12, 16 and 21 post-ovulation. Out of 28 buffaloes, 11 (39.3%) were diagnosed pregnant on day 40 post-ovulation. Retrospective analysis revealed that the buffaloes getting pregnant had larger (p < 0.05) POF diameter. In fact, all the buffaloes (n = 5/5) having POF diameter between >14 and 16 mm conceived, whereas, conception rate in buffaloes with POF diameter between >12 and ≤14 mm (n = 6/17) or <12 mm (n = 0/6) was 35.3% and 0.0%, respectively. A positive correlation (r = 0.57, p < 0.05) was observed between POF diameter and plasma estradiol concentration at estrus. On day 5 post-ovulation, luteal profile was positively correlated (CL: r = 0.34, p < 0.05; plasma progesterone concentration: r = 0.27, p > 0.05) with POF diameter. Further, on the same day, plasma progesterone concentration was positively correlated (r = 0.47, p < 0.05) with CL diameter, however, this correlation was absent (r = 0.05, p > 0.05) during the subsequent luteal phase. Nevertheless, the post-ovulation luteal profile of pregnant buffaloes was higher (p < 0.05) compared to non-pregnant counterparts. In conclusion, the diameter of POF in buffaloes has positive impact on plasma estradiol concentration at estrus, post-ovulation luteal profile and conception rate. The diameter of CL can be used as an indicator of luteal function at early but not at mid or late luteal phase of estrus cycle in buffaloes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author at: Department of Teaching Veterinary Clinical Complex, College of Veterinary Science, CCS Haryana Agriculture University, Hisar 125004, Haryana, India. Tel.: +91 93553 10011. E-mail address: [email protected] (A.K. Pandey). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.12.003
Embryonic mortality is one of the predominant cause for repeat breeding in dairy animals (Diskin and Morris, 2008; Santos et al., 2004). In fact, loses due to early or late (<25 or >25 to 45 days after fertilization, respectively) embryonic mortality could range between 20 to 44 and 8 to 17 per cent, respectively (Humblot, 2001). Endocrine imbal-
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ance, particularly of progesterone and estrogen, is a major reason underlying early embryonic mortality (Diskin and Morris, 2008; Morgan and Lean, 1993; Stevenson et al., 1990). At estrus, pre-ovulatory follicle (POF) is a prime candidate behind endocrine imbalance because POF produces substantial amounts of estradiol and a positive correlation exists between the diameter of POF and plasma estradiol concentration (Noseir, 2003; Perry et al., 2007). Moreover, in dairy cattle studies, it is speculated that POF diameter is important for the subsequent development of corpus luteum (CL) and, hence conception rate. A larger POF may generate a larger CL that will secrete more progesterone and hereby have a positive effect on pregnancy recognition and pregnancy rates (Binelli et al., 2009; Busch et al., 2008). On the contrary, others have reported absence of correlation (Colazo et al., 2009) or negative correlation (Lynch et al., 2010; Vasconcelos et al., 1999) between POF diameter and pregnancy outcome. The secretion of progesterone during early luteal phase is essential for successful establishment of pregnancy. Low plasma progesterone concentration during the early luteal phase was shown in non-pregnant buffaloes compared to their pregnant counterparts (Kavani et al., 2005). It is often predicted that a large CL will release more progesterone in the circulation (Binelli et al., 2009), but the results are not always consistent (Childs et al., 2008). The weight of CL and plasma progesterone concentration is strongly allied on day 5 (Green et al., 2005; Robinson et al., 2005), however, this correlation was lost later on (Robinson et al., 2005). The impact of POF diameter on subsequent luteal profile (CL diameter and plasma progesterone concentration) and conception rate is not established in buffaloes. Moreover, it is not clear whether the diameter of CL is a good indicator of progesterone profile in buffaloes. Therefore, the objectives of present investigation were: (1) to examine the correlation of POF diameter with plasma estradiol concentration on the day of estrus and subsequent conception rate, (2) to investigate the impact of POF diameter on subsequent luteal profile (CL diameter and plasma progesterone concentration) and, (3) to determine the correlation between CL diameter and plasma progesterone concentration.
2. Materials and methods 2.1. Animals and management The study was conducted on 28 reproductively normal Murrah buffaloes. The selected buffaloes (parity: 2–4, body condition score: 4–5 and body weight: 400–500 kg) were regular cycling and were free from any apparent pathological problem of genital tract. Buffaloes were fed chaffed green fodder, wheat straw, concentrates (maize or wheat 60%, groundnut cake 25%, wheat bran 10%, rice bran 5% and common salt 1%), mineral mixture and ad libitum drinking water. All the buffaloes were housed under semi-loose housing system. Lactating buffaloes were milked twice a day, morning (04:00 a.m.) and evening (03:00 p.m.).
2.2. Estrus synchronization and artificial insemination The buffaloes were estrus synchronized using double prostaglandin F2␣ (PGF2␣ ) protocol. In this protocol, buffaloes were administered (i.m.) two injections of a synthetic PGF2␣ analogue (500 g, Cloprostenol Sodium, VetmateTM , Vetcare, Bangalore, India) about 11 days apart. After 48 h of second-PGF2␣ injection, signs of inducedestrus were observed by teaser bull. Bull was paraded once during early morning and once during late evening. First artificial insemination (AI) was carried out during mid to late estrus, and subsequent inseminations were carried out at 24 h interval (Fig. 1). Third AI was done only if dominant ovulatory follicle was detectable by transrectal ultrasonography. Artificial insemination was done using frozen-thawed semen. The straws of frozen semen (French medium straws, 0.5 ml, 40 million sperms per straw) were procured from the university semen bank and all the straws were from the same progeny tested healthy bull. Just before each AI, a straw was thawed using standard thawing protocol. All the inseminations were carried out by the same person after properly checking the microscopic quality of semen. 2.3. Ultrasonographic examinations and blood collection Per-rectal ovarian ultrasonography was carried out using a battery operated B-mode ultrasound scanner (Agroscan AL, ECM, Angouleme, France) equipped with inbuilt interchangeable 5/7.5 MHz linear-array rectal transducer (ALR 575 probe, ECM, Angouleme, France). Scanning was carried out starting from the day of onset of estrus at 24 h interval till ovulation and thereafter, on days 5, 12, 16, 21 post-ovulation (Fig. 1). Ovaries were systematically scanned and images were recorded in the recorder and on a diagram of the ovary by carefully sketching the size and relative location of all follicles and relative location of visible CL (Ghuman et al., 2010). Optimal scan images were frozen and the size of the follicles/CL was determined by measurement of the largest and smallest diameter of the follicles/CL and thereafter, average diameter calculated. All measurements were made using the built-in, on-screen calipers. The follicle which disappeared after the end of estrus was considered as the ovulatory follicle. The ovulatory follicle diameter was, retrospectively, the diameter recorded on the day just before the day of disappearance (ovulation) of follicle. Day of ovulation (day 0) was the last day when the follicle was found intact before disappearing at subsequent examination 24 h later (Ghuman et al., 2010). Pregnancy was confirmed on day 40 post-ovulation by ultrasonography. Blood was collected (10 ml) from the jugular vein in a heparinized vaccutainer vials after each ultrasonography. Plasma was separated immediately after blood collection by centrifugation at 1500 × g for 15 min at 4 ◦ C and plasma aliquots were stored at −20 ◦ C until analysis. 2.4. Hormone assays Plasma estradiol-17 was estimated using a commercially available Direct Immunoenzymatic Assay Kit
A.K. Pandey et al. / Animal Reproduction Science 123 (2011) 169–174
171
Days post- ovulation
Day of ovulation (d0) Days -15
1st PG
-4
-3
2nd PG
-2 -1/0
0
12
5
16
21
40
AI
PD Ultrasonography and blood sampling Fig. 1. Schematic diagram for experiment schedule in buffalo. (AI: artificial insemination; d: day; PD: pregnancy diagnosis; PG: prostaglandin).
(Equipar Diagnostic Company, Italy). Plasma was not extracted in the assay procedure. Anti-estradiol IgG coated 96-well plate was used and 25 l samples/standards were pipetted to the respective wells in duplicate. Thereafter, 100 l conjugate was added in all the wells and the plate was incubated at 37 ◦ C for 120 min. All the wells were washed 4 times with 300 l distilled water. Following addition of substrate solution (100 l), plate was again incubated in dark at room temperature for 30 min. Thereafter, stop solution (100 l) was added to the wells and absorbance of each well was taken at 450 nm within 30 min. The values were calculated by plotting standard curve. The accuracy of assay was 96.7% ± 3.1%. Plasma progesterone concentration was estimated by solid-phase Radioimmunoassay (RIA) procedure. Plasma was not extracted in the assay procedure. Phosphate buffer saline (PBS; 100 l) was added to the numbered glass test tubes to be used for either standards or samples. Progesterone standards/samples were pipetted (100 l) in duplicate. Polyclonal progesterone antiserum raised in the departmental RIA laboratory was used at 1:1000 dilutions (Ghuman et al., 2009). About 100 l of antiserum was added to all the tubes except non-specific binding tube followed by addition of tritiated progesterone (100 l; 10,000 counts per minute) (Amersham Biosciences, UK). Overnight incubation was carried out at 4 ◦ C. Assay was terminated by adding 500 l activated charcoal suspension (Charcoal 0.5 g + Dextran T-70, 0.05 g in 100 ml PBS, pH 7.0) to all the tubes. Tubes were incubated for 10 min at 4 ◦ C. Thereafter, all the tubes were centrifuged at 3000 × g for 10 min. The supernatant containing the antiserum-bound hormone was decanted directly into scintillation vials containing 5 ml scintillation fluid. Radioactivity was determined as counts per minute for one min in the Beckman-6500 multipurpose scintillation counter. The mean intra- and inter-assay coefficients of variance were 6.2 and 9.5 percent, respectively. The sensitivity of progesterone assay was 0.1 ng/ml.
2.5. Statistical analysis Numerical data are represented as mean ± SE. Student’s t-test was employed to compare the POF diameter, plasma estradiol concentration at estrus and post-ovulation luteal profile (CL diameter and plasma progesterone concentration) of pregnant and non-pregnant buffaloes. Correlation coefficient was analyzed between: (a) POF diameter and plasma estradiol concentration at estrus, (b) POF and CL
diameter on day 5, and (c) CL diameter and plasma progesterone concentration on days 5 and 12 post-ovulation. Conception rate was defined as the number of buffaloes diagnosed pregnant on day 40 post-ovulation divided by the number of buffaloes inseminated. Effect of diameterbased categorization of the POF on conception rate was determined by Chi-square analysis. Differences at a p value less than 5% (p < 0.05) was considered statistically significant. All statistical analysis was performed using the SPSS (16.0) system for windows. 3. Results All buffaloes responded to the estrus synchronization treatment and showed estrus signs at 53.8 ± 1.3 h after second-PGF2␣ injection. 3.1. Pre-ovulatory follicle diameter on the day of estrus and conception rate Eleven (39.29%) buffaloes were diagnosed pregnant on day 40 post-ovulation. The mean diameter of POF on the day of estrus was larger (p < 0.05) in buffaloes which ultimately were diagnosed pregnant compared to their non-pregnant counterparts (14.1 ± 0.3 vs 12.2 ± 0.3 mm, respectively; Table 1). Further analysis of conception rate data was carried out based upon the categorization of POF diameter. The conception rate in buffaloes having POF Table 1 Ovarian and endocrine parameters in buffaloes (n = 28) that were diagnosed pregnant or non-pregnant. Parameters
Pregnant (n = 11)
Non-pregnant (n = 17)
Day of estrus Pre-ovulatory follicle diameter, mm 14.1 ± 0.3* Plasma estradiol, pg/ml 35.1 ± 3.2* Plasma progesterone, ng/ml 0.33 ± 0.1
12.2 ± 0.3 22.5 ± 2.5 0.35 ± 0.1
Corpus luteum diameter, mm Day 5 post-ovulation Day 12 Day 16 Day 21
15.7 ± 0.7* 14.6 ± 1.3 15.3 ± 1.3 14.2 ± 0.9
13.3 ± 0.4 14.5 ± 0.7 14.6 ± 0.9 14.0 ± 1.1
Plasma progesterone, ng/ml Day 5 post-ovulation Day 12 Day 16 Day 21
0.87 ± 0.1* 1.56 ± 0.1* 1.92 ± 0.2* 2.1 ± 0.2*
0.52 ± 0.1 0.54 ± 0.1 0.70 ± 0.2 1.1 ± 0.3
*
p < 0.05: within a row.
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diameter on the day of estrus between 10 to ≤12, >12 to ≤14 and >14 to 16 mm was 0%, 35.3% and 100% (2 = 11.72, p < 0.05), respectively. 3.2. Pre-ovulatory follicle diameter, plasma estradiol/progesterone concentration on the day of estrus and pregnancy A positive correlation was observed between the POF diameter and plasma estradiol concentration (r = 0.57, p < 0.05). Retrospective analysis revealed higher (p < 0.05) plasma estradiol concentration on the day of estrus in buffaloes that ultimately conceived as compared to their nonconceiving counterparts (35.1 ± 3.2 vs 22.5 ± 2.5 pg/ml, respectively; Table 1). Plasma progesterone concentration on the day of estrus was similar (p > 0.05) in buffaloes that subsequently conceived or failed to conceive (0.33± 0.1 vs 0.35 ± 0.1 ng/ml, respectively, Table 1). 3.3. Pre-ovulatory follicle diameter, subsequent luteal profiles (CL diameter and plasma progesterone concentration) and pregnancy On day 5 post-ovulation, luteal profile was positively correlated (CL: r = 0.34, p < 0.05; progesterone concentration: r = 0.27, p > 0.05) with POF diameter. In addition, on day 5 post-ovulation, a positive correlation (r = 0.47, p < 0.05) was observed between CL diameter and plasma progesterone concentration, however this correlation was absent on day 12 (r = 0.05, p > 0.05). Also, retrospective analysis revealed that on day 5 post-ovulation, the pregnant buffaloes had higher (p < 0.05, Table 1) post-ovulation luteal profiles (CL diameter and plasma progesterone concentration) than their non-pregnant counterparts. However, on days 12, 16 and 21 post-ovulation, the difference (p < 0.05) between pregnant and non-pregnant buffaloes were observed only in plasma progesterone concentration (Table 1). Unexpectedly, on day 21 post-ovulation, plasma progesterone concentration in non-pregnant buffaloes was found 1.1 ± 0.3 ng/ml (Table 1). Analysis of individual buffalo data (except five buffaloes, those returned to estrus and exhibited estrus signs) revealed that four buffaloes had >1 ng/ml (2.2 ± 0.2 ng/ml) and eight buffaloes had <1 ng/ml (0.5 ± 0.1 ng/ml) plasma progesterone concentration on day 21 post-ovulation. Moreover, CL diameter was also larger (p < 0.05) in former compared to later (16.9 ± 1.9 vs 12.5 ± 0.9 mm, respectively). 4. Discussion This study investigated the relationship of POF diameter with plasma estradiol concentration, post-ovulation luteal profile (CL diameter and plasma progesterone concentration) and conception rate in dairy buffalo. The conception rate was positively related with POF diameter on the day of estrus. Analysis on the basis of diameter-based categorization of the POFs strengthened the findings that larger POF diameter is more competent to establish pregnancy in buffaloes. Several reports in dairy cattle have also proposed positive association between POF diameter and subsequent
conception rate (Bello et al., 2006; Lopes et al., 2007; Mapletoft et al., 2005). Moreover, GnRH-induced ovulation of 14.5 mm diameter follicle was more competent to establish pregnancy compared to a follicle of 10.3 mm (Perry et al., 2005). In contrast, an inverse relationship was also observed between POF diameter and embryo survival (Lynch et al., 2010; Vasconcelos et al., 1999). Higher embryo survival following ovulation of a large POF is due to formation of a large CL which secretes more progesterone. The latter is a major factor responsible for establishment of early embryo in the uterus (Vasconcelos et al., 2001). In dairy cattle, at the time of estrus, the POF is known for increasing circulating concentrations of estradiol and a positive correlation exists between POF diameter and plasma estradiol concentration (Lopes et al., 2007; Lynch et al., 2010). Similarly, in the buffaloes of present study, a positive influence of POF diameter was observed on the plasma estradiol concentration. Moreover, the occurrence of higher plasma estradiol concentration on the day of estrus in pregnant buffaloes than their non-pregnant counterparts suggested that steroid biosynthesis by the POF can influence pregnancy establishment. In contrast, studies in dairy cattle have failed to establish association between plasma estradiol concentration on the day of estrus and the chances of subsequent conception (Busch et al., 2008; Perry et al., 2005). In present study, the larger POF observed on the day of estrus yielded a larger CL on day 5 post-ovulation and had positive influence on plasma progesterone concentration. These findings support the theory established by the studies in dairy cattle that a large POF generates a large CL which subsequently leads to higher circulating progesterone (Lopes et al., 2007; Pfeifer et al., 2009). Actually, a few granulosa cells are present in small POFs which may yield fewer luteal cells subsequent to ovulation and hence lower plasma progesterone concentration (Murdoch and Van Kirk, 1998; Smith et al., 1994). On the other hand, a recent study has failed to observe the correlation of POF diameter with the subsequent CL diameter as well as with plasma progesterone concentration on day 7 post-estrus (Lynch et al., 2010). In the current study, a positive correlation was observed between the CL diameter and subsequent plasma progesterone concentration on day 5 and not on day 12 post-ovulation. Correspondingly, a positive correlation between CL diameter and subsequent plasma progesterone was reported on day 5 of luteal phase, however this relationship was lost by day 8 (Mann, 2009; Robinson et al., 2005). Overall, the findings observed in buffaloes and reported in dairy cattle suggest that the CL diameter could be an indicator of CL function at early- but not at mid- or late-luteal phase of estrus cycle. In the pregnant buffaloes, a larger CL diameter observed on day 5 post-ovulation could be due to the presence of large POF at estrus compared to their non-pregnant counterparts. However, from days 12 to 21 post-ovulation, the absence of difference in CL diameter between pregnant and non-pregnant buffaloes suggested that CL diameter during this period is not the indicator of subsequent pregnancy status in buffaloes. Nevertheless, throughout the postovulation luteal phase, the pregnant buffaloes had higher plasma progesterone concentration than non-pregnant
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buffaloes. Similarly, in a previous study, pregnant buffaloes exhibited higher plasma progesterone concentration between days 10 and 20 post-insemination as compared to their counterparts experiencing embryonic mortality (Campanile et al., 2005). Also, dairy cattle that conceived subsequently had higher plasma progesterone during the post-insemination luteal phase compared to their counterparts failing to conceive (Busch et al., 2008; Lopes et al., 2007). Thus, luteal inadequacy or delay in rise of plasma progesterone concentration during post-breeding luteal phase may be responsible for early embryonic mortality in buffaloes (Campanile et al., 2005; Grimard et al., 2006). In fact, majority of the non-pregnant buffaloes in present study experienced early embryonic mortality as indicated by their low luteal profile (CL diameter and plasma progesterone concentration) on day 21 post-ovulation. Similarly, embryonic mortality between days 8 and 16 post-breeding was suggested as a major contributor to reproductive failure (Beltman et al., 2009; Dunne et al., 2000; Sreenan et al., 2001; Thatcher et al., 1995). Some workers based upon the occurrence of high luteal profile (plasma progesterone concentration: >2.0 ng/ml) on day 21 post-breeding in nonpregnant dairy cattle suggested late embryonic mortality as a cause of pregnancy failure and further hypothesized that pregnancy recognition may have initiated in these cattle, yet these animals failed to maintain pregnancy until day 40 post-breeding (Larson et al., 1997). 5. Conclusion On the day of estrus/breeding, the POF diameter was positively related with plasma estradiol concentration and subsequent conception rate in Murrah buffaloes. The larger POF give way to a larger CL on day 5 post-ovulation. A positive correlation existed between the CL diameter and plasma progesterone concentration on day 5 post-ovulation. Compared to non-pregnant buffaloes, the pregnant buffaloes had a larger CL only on day 5 postovulation, whereas, plasma progesterone concentration was high throughout the post-ovulation luteal phase. Acknowledgements This work was carried out under the project entitled “Antiluteolytic strategies—a novel approach to enhance fertility in buffalo”, financially supported by “National Fund for Basic and Strategic Research in Agricultural Sciences”. Authors are deeply grateful to Dr. J.B. Phogat and Dr. Ravi Kumar for their unconditional help to analyze the data. References Bello, N.M., Steibel, J.P., Pursley, J.R., 2006. Optimizing ovulation to first GnRH improved outcomes to each hormonal injection of ovsynch in lactating dairy cows. J. Dairy Sci. 89, 3413–3424. Beltman, M.E., Lonergan, P., Diskin, M.G., Roche, J.F., Crowe, M.A., 2009. Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers. Theriogenology 71, 1173–1179. Binelli, M., Machado, R., Bergamaschi, M.A.C.M., Bertan, C.M., 2009. Manipulation of ovarian and uterine function to increase conception rates in cattle. Anim. Reprod. 6, 125–134.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis) A.K. Pandey a,∗ , G.S. Dhaliwal b , S.P.S. Ghuman a , S.K. Agarwal c a
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India Department of Teaching Veterinary Clinical Complex, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India c Division of Animal Reproduction, Indian Veterinary Research Institute, Bareilly 243122, Uttar Pradesh, India b
a r t i c l e
i n f o
Article history: Received 31 August 2010 Received in revised form 21 November 2010 Accepted 6 December 2010 Available online 14 December 2010 Keywords: Buffalo Estradiol Luteal profile Pre-ovulatory follicle Progesterone
a b s t r a c t The present study was designed to investigate the impact of pre-ovulatory follicle (POF) diameter on the day of estrus on plasma estradiol concentration, subsequent luteal profile (corpus luteum, CL, diameter and plasma progesterone concentration) and conception rate in buffaloes. Twenty-eight buffaloes were synchronized with synthetic analogue of prostaglandin F2␣ (PGF2␣ ) administered 11 days apart. Transrectal ultrasonography and jugular vein blood sampling was carried out on the day of estrus and on days 0 (day of ovulation), 5, 12, 16 and 21 post-ovulation. Out of 28 buffaloes, 11 (39.3%) were diagnosed pregnant on day 40 post-ovulation. Retrospective analysis revealed that the buffaloes getting pregnant had larger (p < 0.05) POF diameter. In fact, all the buffaloes (n = 5/5) having POF diameter between >14 and 16 mm conceived, whereas, conception rate in buffaloes with POF diameter between >12 and ≤14 mm (n = 6/17) or <12 mm (n = 0/6) was 35.3% and 0.0%, respectively. A positive correlation (r = 0.57, p < 0.05) was observed between POF diameter and plasma estradiol concentration at estrus. On day 5 post-ovulation, luteal profile was positively correlated (CL: r = 0.34, p < 0.05; plasma progesterone concentration: r = 0.27, p > 0.05) with POF diameter. Further, on the same day, plasma progesterone concentration was positively correlated (r = 0.47, p < 0.05) with CL diameter, however, this correlation was absent (r = 0.05, p > 0.05) during the subsequent luteal phase. Nevertheless, the post-ovulation luteal profile of pregnant buffaloes was higher (p < 0.05) compared to non-pregnant counterparts. In conclusion, the diameter of POF in buffaloes has positive impact on plasma estradiol concentration at estrus, post-ovulation luteal profile and conception rate. The diameter of CL can be used as an indicator of luteal function at early but not at mid or late luteal phase of estrus cycle in buffaloes. © 2010 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author at: Department of Teaching Veterinary Clinical Complex, College of Veterinary Science, CCS Haryana Agriculture University, Hisar 125004, Haryana, India. Tel.: +91 93553 10011. E-mail address: [email protected] (A.K. Pandey). 0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2010.12.003
Embryonic mortality is one of the predominant cause for repeat breeding in dairy animals (Diskin and Morris, 2008; Santos et al., 2004). In fact, loses due to early or late (<25 or >25 to 45 days after fertilization, respectively) embryonic mortality could range between 20 to 44 and 8 to 17 per cent, respectively (Humblot, 2001). Endocrine imbal-
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ance, particularly of progesterone and estrogen, is a major reason underlying early embryonic mortality (Diskin and Morris, 2008; Morgan and Lean, 1993; Stevenson et al., 1990). At estrus, pre-ovulatory follicle (POF) is a prime candidate behind endocrine imbalance because POF produces substantial amounts of estradiol and a positive correlation exists between the diameter of POF and plasma estradiol concentration (Noseir, 2003; Perry et al., 2007). Moreover, in dairy cattle studies, it is speculated that POF diameter is important for the subsequent development of corpus luteum (CL) and, hence conception rate. A larger POF may generate a larger CL that will secrete more progesterone and hereby have a positive effect on pregnancy recognition and pregnancy rates (Binelli et al., 2009; Busch et al., 2008). On the contrary, others have reported absence of correlation (Colazo et al., 2009) or negative correlation (Lynch et al., 2010; Vasconcelos et al., 1999) between POF diameter and pregnancy outcome. The secretion of progesterone during early luteal phase is essential for successful establishment of pregnancy. Low plasma progesterone concentration during the early luteal phase was shown in non-pregnant buffaloes compared to their pregnant counterparts (Kavani et al., 2005). It is often predicted that a large CL will release more progesterone in the circulation (Binelli et al., 2009), but the results are not always consistent (Childs et al., 2008). The weight of CL and plasma progesterone concentration is strongly allied on day 5 (Green et al., 2005; Robinson et al., 2005), however, this correlation was lost later on (Robinson et al., 2005). The impact of POF diameter on subsequent luteal profile (CL diameter and plasma progesterone concentration) and conception rate is not established in buffaloes. Moreover, it is not clear whether the diameter of CL is a good indicator of progesterone profile in buffaloes. Therefore, the objectives of present investigation were: (1) to examine the correlation of POF diameter with plasma estradiol concentration on the day of estrus and subsequent conception rate, (2) to investigate the impact of POF diameter on subsequent luteal profile (CL diameter and plasma progesterone concentration) and, (3) to determine the correlation between CL diameter and plasma progesterone concentration.
2. Materials and methods 2.1. Animals and management The study was conducted on 28 reproductively normal Murrah buffaloes. The selected buffaloes (parity: 2–4, body condition score: 4–5 and body weight: 400–500 kg) were regular cycling and were free from any apparent pathological problem of genital tract. Buffaloes were fed chaffed green fodder, wheat straw, concentrates (maize or wheat 60%, groundnut cake 25%, wheat bran 10%, rice bran 5% and common salt 1%), mineral mixture and ad libitum drinking water. All the buffaloes were housed under semi-loose housing system. Lactating buffaloes were milked twice a day, morning (04:00 a.m.) and evening (03:00 p.m.).
2.2. Estrus synchronization and artificial insemination The buffaloes were estrus synchronized using double prostaglandin F2␣ (PGF2␣ ) protocol. In this protocol, buffaloes were administered (i.m.) two injections of a synthetic PGF2␣ analogue (500 g, Cloprostenol Sodium, VetmateTM , Vetcare, Bangalore, India) about 11 days apart. After 48 h of second-PGF2␣ injection, signs of inducedestrus were observed by teaser bull. Bull was paraded once during early morning and once during late evening. First artificial insemination (AI) was carried out during mid to late estrus, and subsequent inseminations were carried out at 24 h interval (Fig. 1). Third AI was done only if dominant ovulatory follicle was detectable by transrectal ultrasonography. Artificial insemination was done using frozen-thawed semen. The straws of frozen semen (French medium straws, 0.5 ml, 40 million sperms per straw) were procured from the university semen bank and all the straws were from the same progeny tested healthy bull. Just before each AI, a straw was thawed using standard thawing protocol. All the inseminations were carried out by the same person after properly checking the microscopic quality of semen. 2.3. Ultrasonographic examinations and blood collection Per-rectal ovarian ultrasonography was carried out using a battery operated B-mode ultrasound scanner (Agroscan AL, ECM, Angouleme, France) equipped with inbuilt interchangeable 5/7.5 MHz linear-array rectal transducer (ALR 575 probe, ECM, Angouleme, France). Scanning was carried out starting from the day of onset of estrus at 24 h interval till ovulation and thereafter, on days 5, 12, 16, 21 post-ovulation (Fig. 1). Ovaries were systematically scanned and images were recorded in the recorder and on a diagram of the ovary by carefully sketching the size and relative location of all follicles and relative location of visible CL (Ghuman et al., 2010). Optimal scan images were frozen and the size of the follicles/CL was determined by measurement of the largest and smallest diameter of the follicles/CL and thereafter, average diameter calculated. All measurements were made using the built-in, on-screen calipers. The follicle which disappeared after the end of estrus was considered as the ovulatory follicle. The ovulatory follicle diameter was, retrospectively, the diameter recorded on the day just before the day of disappearance (ovulation) of follicle. Day of ovulation (day 0) was the last day when the follicle was found intact before disappearing at subsequent examination 24 h later (Ghuman et al., 2010). Pregnancy was confirmed on day 40 post-ovulation by ultrasonography. Blood was collected (10 ml) from the jugular vein in a heparinized vaccutainer vials after each ultrasonography. Plasma was separated immediately after blood collection by centrifugation at 1500 × g for 15 min at 4 ◦ C and plasma aliquots were stored at −20 ◦ C until analysis. 2.4. Hormone assays Plasma estradiol-17 was estimated using a commercially available Direct Immunoenzymatic Assay Kit
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Days post- ovulation
Day of ovulation (d0) Days -15
1st PG
-4
-3
2nd PG
-2 -1/0
0
12
5
16
21
40
AI
PD Ultrasonography and blood sampling Fig. 1. Schematic diagram for experiment schedule in buffalo. (AI: artificial insemination; d: day; PD: pregnancy diagnosis; PG: prostaglandin).
(Equipar Diagnostic Company, Italy). Plasma was not extracted in the assay procedure. Anti-estradiol IgG coated 96-well plate was used and 25 l samples/standards were pipetted to the respective wells in duplicate. Thereafter, 100 l conjugate was added in all the wells and the plate was incubated at 37 ◦ C for 120 min. All the wells were washed 4 times with 300 l distilled water. Following addition of substrate solution (100 l), plate was again incubated in dark at room temperature for 30 min. Thereafter, stop solution (100 l) was added to the wells and absorbance of each well was taken at 450 nm within 30 min. The values were calculated by plotting standard curve. The accuracy of assay was 96.7% ± 3.1%. Plasma progesterone concentration was estimated by solid-phase Radioimmunoassay (RIA) procedure. Plasma was not extracted in the assay procedure. Phosphate buffer saline (PBS; 100 l) was added to the numbered glass test tubes to be used for either standards or samples. Progesterone standards/samples were pipetted (100 l) in duplicate. Polyclonal progesterone antiserum raised in the departmental RIA laboratory was used at 1:1000 dilutions (Ghuman et al., 2009). About 100 l of antiserum was added to all the tubes except non-specific binding tube followed by addition of tritiated progesterone (100 l; 10,000 counts per minute) (Amersham Biosciences, UK). Overnight incubation was carried out at 4 ◦ C. Assay was terminated by adding 500 l activated charcoal suspension (Charcoal 0.5 g + Dextran T-70, 0.05 g in 100 ml PBS, pH 7.0) to all the tubes. Tubes were incubated for 10 min at 4 ◦ C. Thereafter, all the tubes were centrifuged at 3000 × g for 10 min. The supernatant containing the antiserum-bound hormone was decanted directly into scintillation vials containing 5 ml scintillation fluid. Radioactivity was determined as counts per minute for one min in the Beckman-6500 multipurpose scintillation counter. The mean intra- and inter-assay coefficients of variance were 6.2 and 9.5 percent, respectively. The sensitivity of progesterone assay was 0.1 ng/ml.
2.5. Statistical analysis Numerical data are represented as mean ± SE. Student’s t-test was employed to compare the POF diameter, plasma estradiol concentration at estrus and post-ovulation luteal profile (CL diameter and plasma progesterone concentration) of pregnant and non-pregnant buffaloes. Correlation coefficient was analyzed between: (a) POF diameter and plasma estradiol concentration at estrus, (b) POF and CL
diameter on day 5, and (c) CL diameter and plasma progesterone concentration on days 5 and 12 post-ovulation. Conception rate was defined as the number of buffaloes diagnosed pregnant on day 40 post-ovulation divided by the number of buffaloes inseminated. Effect of diameterbased categorization of the POF on conception rate was determined by Chi-square analysis. Differences at a p value less than 5% (p < 0.05) was considered statistically significant. All statistical analysis was performed using the SPSS (16.0) system for windows. 3. Results All buffaloes responded to the estrus synchronization treatment and showed estrus signs at 53.8 ± 1.3 h after second-PGF2␣ injection. 3.1. Pre-ovulatory follicle diameter on the day of estrus and conception rate Eleven (39.29%) buffaloes were diagnosed pregnant on day 40 post-ovulation. The mean diameter of POF on the day of estrus was larger (p < 0.05) in buffaloes which ultimately were diagnosed pregnant compared to their non-pregnant counterparts (14.1 ± 0.3 vs 12.2 ± 0.3 mm, respectively; Table 1). Further analysis of conception rate data was carried out based upon the categorization of POF diameter. The conception rate in buffaloes having POF Table 1 Ovarian and endocrine parameters in buffaloes (n = 28) that were diagnosed pregnant or non-pregnant. Parameters
Pregnant (n = 11)
Non-pregnant (n = 17)
Day of estrus Pre-ovulatory follicle diameter, mm 14.1 ± 0.3* Plasma estradiol, pg/ml 35.1 ± 3.2* Plasma progesterone, ng/ml 0.33 ± 0.1
12.2 ± 0.3 22.5 ± 2.5 0.35 ± 0.1
Corpus luteum diameter, mm Day 5 post-ovulation Day 12 Day 16 Day 21
15.7 ± 0.7* 14.6 ± 1.3 15.3 ± 1.3 14.2 ± 0.9
13.3 ± 0.4 14.5 ± 0.7 14.6 ± 0.9 14.0 ± 1.1
Plasma progesterone, ng/ml Day 5 post-ovulation Day 12 Day 16 Day 21
0.87 ± 0.1* 1.56 ± 0.1* 1.92 ± 0.2* 2.1 ± 0.2*
0.52 ± 0.1 0.54 ± 0.1 0.70 ± 0.2 1.1 ± 0.3
*
p < 0.05: within a row.
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diameter on the day of estrus between 10 to ≤12, >12 to ≤14 and >14 to 16 mm was 0%, 35.3% and 100% (2 = 11.72, p < 0.05), respectively. 3.2. Pre-ovulatory follicle diameter, plasma estradiol/progesterone concentration on the day of estrus and pregnancy A positive correlation was observed between the POF diameter and plasma estradiol concentration (r = 0.57, p < 0.05). Retrospective analysis revealed higher (p < 0.05) plasma estradiol concentration on the day of estrus in buffaloes that ultimately conceived as compared to their nonconceiving counterparts (35.1 ± 3.2 vs 22.5 ± 2.5 pg/ml, respectively; Table 1). Plasma progesterone concentration on the day of estrus was similar (p > 0.05) in buffaloes that subsequently conceived or failed to conceive (0.33± 0.1 vs 0.35 ± 0.1 ng/ml, respectively, Table 1). 3.3. Pre-ovulatory follicle diameter, subsequent luteal profiles (CL diameter and plasma progesterone concentration) and pregnancy On day 5 post-ovulation, luteal profile was positively correlated (CL: r = 0.34, p < 0.05; progesterone concentration: r = 0.27, p > 0.05) with POF diameter. In addition, on day 5 post-ovulation, a positive correlation (r = 0.47, p < 0.05) was observed between CL diameter and plasma progesterone concentration, however this correlation was absent on day 12 (r = 0.05, p > 0.05). Also, retrospective analysis revealed that on day 5 post-ovulation, the pregnant buffaloes had higher (p < 0.05, Table 1) post-ovulation luteal profiles (CL diameter and plasma progesterone concentration) than their non-pregnant counterparts. However, on days 12, 16 and 21 post-ovulation, the difference (p < 0.05) between pregnant and non-pregnant buffaloes were observed only in plasma progesterone concentration (Table 1). Unexpectedly, on day 21 post-ovulation, plasma progesterone concentration in non-pregnant buffaloes was found 1.1 ± 0.3 ng/ml (Table 1). Analysis of individual buffalo data (except five buffaloes, those returned to estrus and exhibited estrus signs) revealed that four buffaloes had >1 ng/ml (2.2 ± 0.2 ng/ml) and eight buffaloes had <1 ng/ml (0.5 ± 0.1 ng/ml) plasma progesterone concentration on day 21 post-ovulation. Moreover, CL diameter was also larger (p < 0.05) in former compared to later (16.9 ± 1.9 vs 12.5 ± 0.9 mm, respectively). 4. Discussion This study investigated the relationship of POF diameter with plasma estradiol concentration, post-ovulation luteal profile (CL diameter and plasma progesterone concentration) and conception rate in dairy buffalo. The conception rate was positively related with POF diameter on the day of estrus. Analysis on the basis of diameter-based categorization of the POFs strengthened the findings that larger POF diameter is more competent to establish pregnancy in buffaloes. Several reports in dairy cattle have also proposed positive association between POF diameter and subsequent
conception rate (Bello et al., 2006; Lopes et al., 2007; Mapletoft et al., 2005). Moreover, GnRH-induced ovulation of 14.5 mm diameter follicle was more competent to establish pregnancy compared to a follicle of 10.3 mm (Perry et al., 2005). In contrast, an inverse relationship was also observed between POF diameter and embryo survival (Lynch et al., 2010; Vasconcelos et al., 1999). Higher embryo survival following ovulation of a large POF is due to formation of a large CL which secretes more progesterone. The latter is a major factor responsible for establishment of early embryo in the uterus (Vasconcelos et al., 2001). In dairy cattle, at the time of estrus, the POF is known for increasing circulating concentrations of estradiol and a positive correlation exists between POF diameter and plasma estradiol concentration (Lopes et al., 2007; Lynch et al., 2010). Similarly, in the buffaloes of present study, a positive influence of POF diameter was observed on the plasma estradiol concentration. Moreover, the occurrence of higher plasma estradiol concentration on the day of estrus in pregnant buffaloes than their non-pregnant counterparts suggested that steroid biosynthesis by the POF can influence pregnancy establishment. In contrast, studies in dairy cattle have failed to establish association between plasma estradiol concentration on the day of estrus and the chances of subsequent conception (Busch et al., 2008; Perry et al., 2005). In present study, the larger POF observed on the day of estrus yielded a larger CL on day 5 post-ovulation and had positive influence on plasma progesterone concentration. These findings support the theory established by the studies in dairy cattle that a large POF generates a large CL which subsequently leads to higher circulating progesterone (Lopes et al., 2007; Pfeifer et al., 2009). Actually, a few granulosa cells are present in small POFs which may yield fewer luteal cells subsequent to ovulation and hence lower plasma progesterone concentration (Murdoch and Van Kirk, 1998; Smith et al., 1994). On the other hand, a recent study has failed to observe the correlation of POF diameter with the subsequent CL diameter as well as with plasma progesterone concentration on day 7 post-estrus (Lynch et al., 2010). In the current study, a positive correlation was observed between the CL diameter and subsequent plasma progesterone concentration on day 5 and not on day 12 post-ovulation. Correspondingly, a positive correlation between CL diameter and subsequent plasma progesterone was reported on day 5 of luteal phase, however this relationship was lost by day 8 (Mann, 2009; Robinson et al., 2005). Overall, the findings observed in buffaloes and reported in dairy cattle suggest that the CL diameter could be an indicator of CL function at early- but not at mid- or late-luteal phase of estrus cycle. In the pregnant buffaloes, a larger CL diameter observed on day 5 post-ovulation could be due to the presence of large POF at estrus compared to their non-pregnant counterparts. However, from days 12 to 21 post-ovulation, the absence of difference in CL diameter between pregnant and non-pregnant buffaloes suggested that CL diameter during this period is not the indicator of subsequent pregnancy status in buffaloes. Nevertheless, throughout the postovulation luteal phase, the pregnant buffaloes had higher plasma progesterone concentration than non-pregnant
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buffaloes. Similarly, in a previous study, pregnant buffaloes exhibited higher plasma progesterone concentration between days 10 and 20 post-insemination as compared to their counterparts experiencing embryonic mortality (Campanile et al., 2005). Also, dairy cattle that conceived subsequently had higher plasma progesterone during the post-insemination luteal phase compared to their counterparts failing to conceive (Busch et al., 2008; Lopes et al., 2007). Thus, luteal inadequacy or delay in rise of plasma progesterone concentration during post-breeding luteal phase may be responsible for early embryonic mortality in buffaloes (Campanile et al., 2005; Grimard et al., 2006). In fact, majority of the non-pregnant buffaloes in present study experienced early embryonic mortality as indicated by their low luteal profile (CL diameter and plasma progesterone concentration) on day 21 post-ovulation. Similarly, embryonic mortality between days 8 and 16 post-breeding was suggested as a major contributor to reproductive failure (Beltman et al., 2009; Dunne et al., 2000; Sreenan et al., 2001; Thatcher et al., 1995). Some workers based upon the occurrence of high luteal profile (plasma progesterone concentration: >2.0 ng/ml) on day 21 post-breeding in nonpregnant dairy cattle suggested late embryonic mortality as a cause of pregnancy failure and further hypothesized that pregnancy recognition may have initiated in these cattle, yet these animals failed to maintain pregnancy until day 40 post-breeding (Larson et al., 1997). 5. Conclusion On the day of estrus/breeding, the POF diameter was positively related with plasma estradiol concentration and subsequent conception rate in Murrah buffaloes. The larger POF give way to a larger CL on day 5 post-ovulation. A positive correlation existed between the CL diameter and plasma progesterone concentration on day 5 post-ovulation. Compared to non-pregnant buffaloes, the pregnant buffaloes had a larger CL only on day 5 postovulation, whereas, plasma progesterone concentration was high throughout the post-ovulation luteal phase. Acknowledgements This work was carried out under the project entitled “Antiluteolytic strategies—a novel approach to enhance fertility in buffalo”, financially supported by “National Fund for Basic and Strategic Research in Agricultural Sciences”. Authors are deeply grateful to Dr. J.B. Phogat and Dr. Ravi Kumar for their unconditional help to analyze the data. References Bello, N.M., Steibel, J.P., Pursley, J.R., 2006. Optimizing ovulation to first GnRH improved outcomes to each hormonal injection of ovsynch in lactating dairy cows. J. Dairy Sci. 89, 3413–3424. Beltman, M.E., Lonergan, P., Diskin, M.G., Roche, J.F., Crowe, M.A., 2009. Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers. Theriogenology 71, 1173–1179. Binelli, M., Machado, R., Bergamaschi, M.A.C.M., Bertan, C.M., 2009. Manipulation of ovarian and uterine function to increase conception rates in cattle. Anim. Reprod. 6, 125–134.
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