Postpartum (>120 days) true anestrus (n=12) and subestrus (n=13) buffaloes were treated with single i/m injection of Buserelin acetate (GnRH) 10 μg and PGF2α (Cloprostenol) 500 μg, respectively. Eight (66.67%) anestrus and 12 (92.31%) subestrus buffaloes responded to treatment with mean estrus induction intervals of 13.00±1.85 days and 67.67±0.97 hrs, respectively. The overall 3 cycles’ conception rates were 87.50 (7/8) and 75.00 (9/12) per cent with mean number of services per conception 2.28 and 2.55, respectively, in two groups. Mean plasma progesterone (P4) concentrations on the day of treatment and on the day of estrus were at basal level in both conceived and non-conceived animals of anestrus group, but on day 12 post-AI, the levels increased significantly (p<0.01), with higher (p<0.05) values in pregnant as compared to non-pregnant animals. The plasma P4 values on the day of PGF2α injection were high in subestrus animals, which dropped significantly (p<0.01) with onset of behavioural estrus and on day 12 post-AI, the mean levels again increased significantly (p<0.01), and in pregnant buffaloes it was higher (p<0.05) as compared to non-pregnant animals. Protein levels were higher at all stages in non-pregnant than in pregnant animals of anestrus group, but differed significantly on the day of estrus in subestrus animals. Plasma cholesterol levels were higher in pregnant than in non-pregnant animals of anestrus group at each observation, with significant difference recorded on the day of GnRH treatment. Its levels continuously declined significantly (p<0.05) from the day of treatment to estrus and to day 12 post-AI in pregnant animals, suggesting its better utilization in steroidogesis. However, a reverse trend in plasma cholesterol concentrations was noted between pregnant and non-pregnant animals of subestrus group. The cholesterol level was non-significantly higher in subestrus animals than in those of anestrus group. The concentrations of plasma triglycerides in anestrus and subestrus animals were lower in pregnant than in non-pregnant animals. There was no much variation in values of minerals Ca, P, Mg between different days or between pregnancy status in any of the infertile groups. In general, the hormonal therapy was beneficial in improving fertility in anestrus and subestrus buffaloes and suitably altered the plasma progesterone profile.
Buffalo is the premier dairy animal in the developing countries of Asia and it is the mainstay of the Indian dairy industry, contributing over 60 per cent of the total milk production (Mondal et al., 2010). Postpartum fertility is a major factor of economic importance in buffalo reproduction. The nutritional, management and environmental factors have an adverse impact on postpartum fertility. Maintenance of desirable calving interval is dependent on the buffaloes’ ability to conceive within the shortest possible time after parturition, but is seldom achieved. Optimum nutrition and use of hormonal regimes, viz., GnRH in anestrus and PGF2α in subestrus buffaloes can be helpful in obtaining good conception rate with less number of services per conception, short service period and calving interval (Khasatiya et al., 2004, 2006, Butani et al., 2014). The present study was therefore, aimed to evaluate the estrus response, conception rate and blood plasma progesterone and biochemical profile of postpartum anestrus and subestrus buffaloes treated once with GnRH and PGF2α under field conditions.
Materials and Methods
The investigation was carried out during winter season from January to March 2016 in the milk shed areas of Panchamrut Dairy, Godhra, Gujarat. Anestrus (n=12) and subestrus (n=13) buffaloes beyond 120 days postpartum, and confirmed by examination per rectum twice 10 days apart, were treated with Buserelin acetate @ 10 μg i/m (GnRH, Inj. Receptal, 2.5 ml, MSD) and Cloprostenol @ 500 μg i/m (PGF2α, Inj. Estrumet, 2 ml, MSD), respectively. The estrus response, estrus induction interval and conception rates were recorded for animals of both the groups. Animals detected in estrus were inseminated by village AI workers. The animals were observed for return to estrus up to three cycles and returning animals were bred through AI. Pregnancy was confirmed by examination per rectum 60 days post-AI.
Blood samples were collected from the representative animals (n=6 and 9) on the day of treatment, day of induced estrus/AI and day 12 post-AI by jugular vein puncture in heparinized vacutainers. The plasma was separated out by centrifugation of samples on the spot and stored at -20°C with a drop of merthiolate (0.1%) as preservative until analyzed. The plasma progesterone was estimated by employing standard RIA technique of Kubasic et al. (1984). The levels of protein, cholesterol, triglycerides and macro-minerals (Ca, P, Mg) were estimated by using standard procedures and assay kits of Coral Clinical System, Goa on biochemistry analyzer. The generated data were analyzed statistically using Chi square test for percent conception rates, and CRD and DMRT for blood profile on SPSS software version 20.00 (Snedecor and Cochran 1994).
Results and Discussion
Effect of GnRH and PGF2α Therapy
Among anestrus buffaloes (n=12) treated with GnRH, 8 (66.67%) animals responded to treatment with mean estrus induction interval of 13.00±1.85 days. The conception rates at first, second and third service were recorded as 25.00, 66.67 and 50.00 per cent, respectively, with overall of 3 cycles as 87.50 (7/8) per cent and mean number of services per conception being 2.28. Among PGF2α treated subestrus buffaloes (n=13), 12 (92.31%) animals responded to treatment with mean estrus induction interval of 67.67±0.97 hrs. The conception rates at first, second and third service were 41.67, 42.86 and 25.00 percent, respectively (Table 1) with overall of 3 cycles as 75.00 % (9/12) percent and mean number of services per conception being 2.55.
Table 1: Estrus induction response, estrus induction interval and conception rates of anestrus/subestrus buffaloes to GnRH/PGF2α treatment
|Group & Treatment||Estrus response to treatment||Estrus induction interval following treatment||Conception rate (%) among responded ones||No. of services / conception|
|1st cycle||2nd cycle||3rd cycle||Overall|
|Anestrus, GnRH 10 µg (n=12)||8 (66.67%)||13.00±1.85(days)||25.00
|Subestrus, PGF2α 500 µg (n=13)||12 (92.31%)||67.67±0.97 hr||41.67 (5/12)||42.86 (3/7)||25.00
Considerable variations in rates of estrus induction with GnRH treatment in anestrus animals have been documented. The estrus induction rate (66.67%) recorded in present study corroborated with the previous findings of Khasatiya et al. (2006), Puranik et al. (2010) and Purkayastha et al. (2015); however, higher rate (89.66%) has been reported by Butani et al. (2014). The estrus induction interval observed (13.00±1.85 days) in present study approximated with observations reported by Mohammed et al. (1999) and Puranik et al. (2010). However, a great variation in estrus induction interval with GnRH treatment in anestrus buffaloes have been reported (Thakur et al., 1996, Khasatiya et al., 2004, Butani et al., 2014 and Purkayastha et al., 2015). Two (25.00%) animals conceived at induced estrus among GnRH responded animals (n=8), however, four and one animals conceived at second and third cycles, respectively, giving overall three cycles’ conception rate of 87.50 per cent (Table 1). These findings corroborated with the reported conception rates of 80 to 91 per cent by Khasatiya et al. (2004), Sah and Nakao (2010), and Parmar et al. (2012). However, comparatively lower conception rate of 73.07 to 76.47 per cent has been documented by Puranik et al. (2010) and Butani et al. (2014) and 64.00 per cent by Purkayastha et al. (2015). The estrus induction response (92.31%) observed in PGF2α treated subestrus buffaloes corroborated with previous findings of 85.70 to 94.44 per cent (Pant and Singh 1991, Koli et al., 2005, Honparkhe et al., 2008). However, comparatively lower results to the extent of 75-80 per cent have been documented by others (Kharche and Srivastava 2001; Shukla and Dabas, 2007) and a very low response of 30.77 per cent by Rathod et al. (2015), while Butani et al. (2014) reported 100 per cent estrus induction response following PGF2α treatment in subestrus buffaloes. The mean estrus induction interval observed (67.67±0.97 hrs) was also in line with the findings reported by Pant and Singh (1991) and Butani et al. (2014). The overall conception rate of 75.00 per cent obtained closely corroborated with the report of Butani et al. (2014) as 76.47 per cent, but was comparatively higher than 44 to 65 per cent reported by others (Totewad et al., 2007; Honparkhe et al., 2008).
Effect of Hormone Therapy on Plasma Progesterone
The plasma progesterone concentrations in true anestrus buffaloes were at the basal level just before GnRH treatment without any follicular or luteal function on per rectal palpation. The initial mean level was much lower in conceived than non-conceived animals (0.18±0.08 vs. 0.68±0.22 ng/dl). On day 12 post-AI, the levels of plasma progesterone increased significantly (p<0.01), with higher (p<0.05) values in pregnant as compared to non-pregnant animals (4.88±0.32 vs. 3.17±0.49 ng/ml). The results indicated better luteal efficiency of induced ovulatory estrus in pregnant buffaloes for maintenance of pregnancy (Table 2). The mean plasma progesterone concentration found in subestrus animals on the day of PGF2α injection was higher, as of luteal phase, due to presence of CL on the ovary. The values in animals that became pregnant and remained non-pregnant were 4.33±0.59 and 3.16±0.70 ng/ml . The progesterone levels dropped significantly (p<0.01) within three days following PGF2α injection with onset of behavioural estrus due to luteolysis in all the animals. Mean plasma P4 concentrations increased significantly (p<0.01) on day 12 post-AI as compared to that on day of estrus (3.97±0.39 vs 0.37±0.06 ng/ml), its level being significantly higher (p<0.05) in pregnant than in non-pregnant animals (4.98±0.30 vs. 3.47±0.4 ng/ml). There was no significant difference in progesterone concentration between pregnant and non-pregnant buffaloes on the day of treatment and day of estrus.
Progesterone profile on the day of treatment was significantly (p<0.01) lower in anestrus than in subestrus animals in present study (0.51±0.18 and 3.55±0.52, ng/ml), but it did not vary on other days (Table 2). Similar observations have been reported by Khasatiya et al. (2004) and Butani et al. (2011) in subestrus and anestrus animals.
Table 2: Plasma progesterone, protein, cholesterol and triglycerides profile on day of treatment, day of estrus/AI and day 12 post-AI in hormonally treated conceived and non-conceived anestrus and subestrus buffaloes
|Group/ Treatment||Status||No.||Day of Treatment||Day of Estrus/AI||Day 12
|Plasma Progesterone (ng/ml)|
|Anestrus (GnRH 10 µg)||Pregnant||2||0.18±0.08a||0.15±0.05a||4.88q±0.32b||1.74±1.00|
|Subestrus (PGF2α, 500 µg)||Pregnant||3||4.33±0.59b||0.24±0.02a||4.98q±0.30b||3.19±0.77|
|Plasma Total Protein (g/dl)|
|Anestrus (GnRH 10 µg)||Pregnant||2||7.73±0.33||7.95±0.06||7.89±0.02||7.85±0.09|
|Subestrus (PGF2α, 500 µg)||Pregnant||3||8.18±0.01||8.41q ±0.06||8.28±0.14||8.29q ±0.06|
|Non-prg||6||7.74±0.17||7.94p ±0.17||7.92±0.22||7.86p ±0.10|
|Plasma Total Cholesterol (mg/dl)|
|Anestrus (GnRH 10 µg)||Pregnant||2||77.13q±1.22b||71.61±1.16ab||66.19±1.96a||71.64q±2.11|
|Subestrus (PGF2α, 500 µg)||Pregnant||3||63.70±6.80||66.98±12.21||67.88±6.18||66.19±4.46|
|Plasma Triglycerides (mg/dl)|
|Anestrus (GnRH 10 µg)||Pregnant||2||13.29±1.01||9.95±0.56||14.39±2.59||12.54±1.12|
|Subestrus (PGF2α, 500 µg)||Pregnant||3||14.13±4±.96||11.23±3.64||11.44±2.50||12.27±1.97|
Means with different superscripts differ significantly (P<0.05) between days (a, b, c); between pregnant and non-pregnant groups (p, q) and between overall treatment (m, n).
Effect of Hormone Therapy on Plasma Biochemical Profile
The mean concentrations of plasma total protein in GnRH treated anestrus buffaloes were slightly higher at all stages in non-pregnant than pregnant animals, while in subestrus animals, the mean protein levels were apparently higher in pregnant than in non-pregnant buffaloes with significant (p<0.05) difference on the day of estrus (8.41±0.06 vs 7.94±0.17 g/dl). However, the effect of day was found to be non-significant in both anestrus and subestrus animals (Table 2). The plasma protein concentration recorded was similar to observations as reported by Lodhi et al. (1998) and Shah et al. (2003). Srivastava and Sahni (2000) found higher level of total protein in pregnant than in non-pregnant animals, however, Khasatiya et al. (2006) did not find significant variation in weekly mean plasma protein levels between PGF2α treated conceived and non-conceived subestrus animals. However, others (Kumar et al., 2010, Butani et al., 2011) reported significantly (p<0.05) lower serum total protein in anestrus and/or subestrus animals than cyclic ones.
Mean concentrations of plasma total cholesterol in anestrus animals were apparently higher in pregnant than in non-pregnant animals at all stages, with significant (p<0.05) difference on the day of GnRH treatment (77.13±1.22 vs 58.55±5.59 mg/dl). Moreover, its levels gradually and significantly (p<0.05) declined from the day of treatment to estrus to day 12 post-AI in pregnant animals, suggesting its better utilization in steroidogesis. In subestrus animals, however a reverse trend was noted in total cholesterol concentrations with non-significantly higher values in non-pregnant than in pregnant animals at all stages of observation (Table 2). Plasma total cholesterol level was non-significantly higher in subestrus than in anestrus animals at all stages of observation, which corroborated with earlier reports (Khasatiya et al., 2006, Patel et al., 2007, Ali and Shukla 2012) for cyclic and anestrus buffaloes. However, Butani et al. (2011) reported significantly lower (p<0.01) level of plasma cholesterol in anestrus as compared to subestrus animals. Non-significant differences were recorded in mean concentrations of plasma triglycerides in GnRH treated conceived and non-conceived animals of anestrus group at all stages of observation. Similar trend was observed in animals of subestrus group (Table 2). Contrary to present observation, Khasatiya (2003) reported significantly higher triglycerides level in PGF2α treated conceived subestrus animals than in non-conceived subestrus buffaloes. Patel and Dhami (2005) reported significantly (p<0.05) higher triglycerides levels in GnRH treated conceived than non-conceived HF cows, but their values were 4 times higher (63.19±0.70 vs 60.45±0.99 mg/dl) than the present observations.
Effect of Hormone Therapy on Plasma Minerals Profile
Mean concentrations of plasma calcium did not reveal any consistent trend between days or between pregnant and non-pregnant animals of GnRH treated anestrus and PGF2α treated subestrus buffaloes. Singh et al. (2005) and Chaurasia et al. (2010) reported significantly (p<0.05) lower levels of plasma calcium in anestrus as compared to normal cyclic buffaloes, however Butani et al. (2011) could not find any difference. There was not much variation in mean plasma inorganic phosphorus concentrations between different days or between pregnancy status in any of the groups. However, Butani et al. (2009) documented significantly higher (P<0.01) inorganic phosphorus in PGF2α treated subestrus as compared to GnRH treated anestrus buffaloes. The trend of plasma inorganic phosphorus in anestrus and subestrus buffaloes observed in the present study is in accordance with Shah et al. (2003). Higher level of inorganic phosphorus has been reported in cyclic than in acyclic animals by several researchers (Singh et al., 2005, Patel et al., 2007, Churasia et al., 2010, Kumar et al., 2010 and Jayachandran et al., 2013).
Mean concentrations of plasma magnesium also did not vary between different days or between pregnancy status in any of two groups. Plasma magnesium concentrations in GnRH treated pregnant animals (2.76±0.15, 2.65±0.23 and 2.79±0.25 mg/dl) were insignificantly higher as compared to non-pregnant animals (2.46±0.12, 2.60±0.12 and 2.67±0.08 mg/dl) on the day of each observation, but no such trend was noted in PGF2α treated pregnant and non-pregnant subestrus animals. Butani et al. (2009) reported slightly higher magnesium in conceived as compared to non-conceived animals.
The present findings in general depicted beneficial effect of use of GnRH in postpartum anestrus and PGF2α in subestrus buffaloes for improving estrus expression and fertility without affecting blood biochemical constituents, except the plasma progesterone and cholesterol concentrations.
We thank the Dean of the Veterinary faculty, the farmers and inseminators of concerned villages of Panchamrut Dairy, and Research Scientist & Head, RBRU, Anand for the financial support and kind cooperation extended in field and lab work of this study.