Ovarian dynamics and conception rate
The ovarian response to Double Ovsynch protocol in post-pubertal acyclic heifers and postpartum acyclic buffaloes is presented in Table 1. Acyclic Jaffarabadi animals that ovulated in response to injection GnRH-1 were 66.66, 33.33 and 33.33 per cent, respectively, in group I, II and III. However, only 50.0 per cent animals in group I and none in group II and III ovulated in response to injection GnRH-2 on day 10. On GnRH-3 injection (day 17) 33.33 per cent animals in group-I and 83.33 per cent each in group-II and III ovulated and in response to injection GnRH-4 (day 26) 83.33 per cent animals each in group I and II and 66.66 per cent in group III ovulated (Table 1).
In group I, 5 (83.33%) animals maintained CL till day 21 post-FTAI. However, one animal failed to maintain CL on day 35 post-FTAI and was found non-pregnant on day 70. In group-II, all 5 (83.33%) animals maintained CL till day 35 post-FTAT. However in group III, only one animal could maintain CL on day 35-FTAI. The first service/FTAI conception rate in double Ovsynch treated animals was the highest in group II (83.33%) followed by group I (66.66%) and the least in group III (16.66%) (Table 1).
Efficiency of ovulation synchronization protocol depends on the ovarian response to the first GnRH injection and it is recognized as an important factor in synchronization of the ovulations for FTAI (De
Jarnette et al., 2001). Compared to present trend and ovulation rates observed in Jaffarabadi buffalo heifers,
Hoque et al., (2014) reported higher ovulation rate with the Ovsynch treatment (83.3%) in buffaloes, while
Cirit et al., (2007) recorded the ovulation rate of 88.9 and 94.5% after the first and second GnRH treatment in cattle with the same protocol. In the present study on acyclic animals, more heifers became pregnant (Group-I 66.66% and Gr-II, 83.33%) than multiparous buffaloes (Gr-III, 16.66%), which seemed to be mostly due to higher fertility in primiparous rather than multiparous animals. It is generally accepted that primiparous cows are more likely to be anovular compared with multiparous cows
(Silva et al., 2007) and respond well to hormone therapy. Greater circulating plasma progesterone during follicular development during double Ovsynch could decrease LH pulsatility possibly improving the competency of the dominant follicle and/or the quality of the ovulated oocyte
(Silva et al., 2007). Souza et al., (2008) and Astiz and Fargas (2013) reported greater P/AI in primiparous cows subjected to double Ovsynch than those of other protocols.
Dirandeh et al., (2015) also reported that double Ovsynch increased pregnancy per AI (P/AI) only in primiparous (65.2 vs 45.2%) as compared to multiparous (37.5 vs 39.3%) cows. Further, significantly more number of cows in the double Ovsynch protocol had CL at first GnRH, greater ovulatory response to second GnRH injection, larger mean diameter of ovulatory follicle at timed AI (TAI) and greater percentage of pregnancy at 32 days after AI compared with other synchronized cows with Presynch-GnRH-Ovsynch and Presynch-Ovsynch
(Dirandeh et al., 2015).
The success of ovulation synchronization protocol has been related to ovulatory response to first GnRH injection of Ovsynch protocol
(Vasconcelos et al., 1999) and progesterone concentration prior to PGF
2α injection
(Martins et al., 2011). A lack of follicle turnover due to failure to respond to the initial GnRH administration might compromise the quality of embryos and consequently reduce P/AI. The failure of certain animals to conceive at induced estrus/ovulation is of interest. Low conception rate in group-III may be due to deficient CL function or lack of ovulatory response to GnRH injections due to multi-parity of buffaloes. In all probability this was primarily a consequence of embryonic mortality after maternal recognition of pregnancy since all animals had well developed corpus luteum on day 12 post-AI (Table 1). Moreover, during early lactation, reproductive performance of dairy animals, particularly conception rate, may be negatively associated with the magnitude of the negative energy balance (Neble and McGilliard, 1993).
The present anestrus buffalo possibly would have responded to the first GnRH injection, but the PGF
2α induced incomplete luteolysis might have resulted in a persistent follicle (non-ovulatory)
(Twagiramungu et al., 1994) that would have ovulated following administration of the second dose of GnRH and LH surge
(Wiltbank et al., 1996). Incomplete luteal regression has been reported in lactating dairy cows receiving ovsynch, with 7% of cows exhibiting high (>2.0 ng/ml) progesterone, 48 h after PGF
2α treatment
(Moreira et al., 2001). However, the multiparous buffaloes in the present study did not conceive. The possible retention of the Graafian follicle for an extended period might have led to damage of the oocyte to such an extent that even inseminating close to the time of ovulation might not have ensured conception
(Duchens et al., 1994). It is also possible that some cows receiving Ovsynch ovulated smaller follicles due to administration of GnRH when compared with cows ovulating after a spontaneous estrus after acquisition of ovulatory capacity, but before the final stages of follicular maturity. Ovulation of smaller follicles might result in smaller corpus luteum that produce less progesterone
(Vasconcelos et al., 1999) and perhaps exhibit a delayed responsiveness to PGF
2α due to expression of fewer PGF
2α receptors.
Follicle Population and Size in Double Ovsynch Treated Animals
Ultrasound monitoring of ovaries of animals from group I revealed significantly increased (
p<0.05) numbers of small follicles on day 24 (4.10±0.33) and 26 (4.50±0.22) as compared to those of previous days (2.60±0.19 to 3.40±0.19). Similarly, the average number of small follicles in group II also differed significantly, with the lowest value on day 7 (2.60±0.19) and the highest on day 24 (3.90±0.67). However, in animals of group III it did not show any variation over the periods (3.10±0.19 to 3.50±0.22). The effect of synchronization treatment on number of medium sized follicles was however non-significant in all three groups. Further, the average number of large sized follicles was also non-significantly different in group I and II, though the mean numbers increased with repeated hormone therapy. However, in group III on day 26 (2.20±0.25) it was the highest, though at par with that on days 10, 17 and 24 and differed significantly (
p<0.05) on day 0 and day 7 (1.00±0.27 and 0.60±0.19) (Table 2). These observations imply that repeated GnRH and PG injections caused synchronized growth, ovulation and re-growth of dominant follicles/CLs in double Ovsynch treated animals particularly postpartum buffaloes. The total number of follicles in acyclic post-pubertal Jaffarabadi heifers of group I under double Ovsynch treatment increased gradually over the period of study with significantly (
p<0.01) higher value on day 26 (9.00±0.26) as compared to day 0 and day 7 (6.10±0.29 and 6.60±0.62). Total number of follicles in group II and III also revealed similar trend, but the differences in mean values were statistically non-significant (Table 3). Moreover, the mean number of small, medium and large and total follicles did not differ significantly between groups during any of the study periods (Table 2, 3).
The mean diameter of large follicle in animals of group I, II and III gradually increased with repeated hormone therapy of the protocol, but the differences between periods were significant (
p<0.001) only in acyclic buffaloes of group III, with the highest diameter on day 17 (12.53±0.85 mm) and day 26 (12.15±0.73 mm) as compared to that observed on day 7 (8.84±0.71 mm). The diameters of large follicles were little larger at most periods in buffaloes of group-III as compared to younger heifers of group-I. The average diameters of subordinate follicles in animals of all three groups followed the same trends as those of large follicles and the differences were significant (
p<0.001) only in group-III with the largest diameter on day 26 (10.72±1.00 mm) and smallest on day 7 (6.62±0.48 mm) of treatment. Moreover, the mean diameters of subordinate follicles in all three groups of double Ovsynch treatment were statistically similar at each period of study, although the diameter of large follicle was maximum in group-III and that of subordinate follicle was lower in group-II on day 17, while both were lower on day 26 compared to other groups (Table 3).
The mean numbers of total follicles visualized on surface of buffalo ovaries in the present study were greater than those reported by
Kumar et al., (1997). Similar trend in terms of total number of follicles and diameter of ovulatory follicle as well as subordinate follicle were also reported in Murrah buffaloes by
Pottupenjera et al., (2018). The mean diameters of large follicles in the present study were also close to the previous reports of
Derar et al., (2012) and
Jerome et al., (2016). However, some researchers
(Baruselli et al., 1997; Barile et al., 2007; Yindee et al., 2011) reported larger mean diameters of large follicles than the present values. These variations could be due to variations in breed of animals, cyclical/ acyclic and nutritional status of animals, climatic conditions, season, potency of preparations used
etc. In the present investigation, irrespective of groups, the maximum diameter of large/dominant follicle, however, coincided with day of FTAI and it was ovulatory one as confirmed by presence of CL on day 12 post-AI in most of the animals.
Hormonal profile
The levels of FSH varied from 4.87±0.54 to 13.09±1.11 ng/ml during different periods of treatment among three groups of animals. The values were significantly (
p<0.001) higher on most of the sampling days in postpartum buffaloes of group-III as compared to those in post-pubertal heifers of group-I and II. Further, the plasma FSH levels fluctuated among all three groups with significant differences in heifers between sampling days mostly in accordance with the hormone GnRH injected, which triggered the release of FSH from pituitary gland. The effect of 2
nd and 3
rd GnRH injection was more pronounced in heifers and 4
th injection in buffaloes. The higher levels of FSH on day 12 and 35 post-AI in buffaloes of group-III were associated with non-pregnancy and repeat cycles, while most heifers had conceived at FTAI and hence low or basal levels of FSH were noted on these days (Table 4). The pattern of plasma FSH profile observed in treated animals concurred well with the physiological changes in the ovaries and agreed with the earlier reports
(Singh et al., 2001; Mondal et al., 2004). The life span of the first dominant follicle has also been correlated to the FSH surge of the estrous cycle and to the emergence of a new follicular wave
(Adams et al., 1992). Studies have established FSH as the key factor in determining the follicular wave pattern in cattle
(Adams et al., 1994). The present values of FSH however were much lower than those reported by
Singh et al., (2001) during estrus, but higher than those reported by
Mondal et al., (2004).
The mean levels of plasma LH in animals under study varied from 0.36±0.03 to 1.56±0.37 ng/ml on different days of treatment across groups. The values of plasma LH clearly fluctuated in heifers of both group-I and II between periods with statistically significant differences in Group-I. This was according to the GnRH/PG therapy and pituitary-ovarian response. Moreover, the levels from day 10 to day 27 were significantly (
p<0.001) higher in responding heifers than the corresponding values of non-responding acyclic buffaloes (Table 4). These findings on plasma levels of LH coincided well with the previous report of Batra and Pandey (1983). However, Roy and Prakash (2008) recorded much higher values of LH than the present findings. According to
Arthur et al., (1989) growth of both germinal and endocrine components of ovary requires only the basal secretion of LH. Thus in present study, the ovarian stimulation noticed in terms of follicular and luteal structures following estrus synchronization protocol with the reported levels is justified, since these elevations/peaks were followed by ovulation in most of the treated heifers. Moreover, the absolute plasma levels of LH/FSH depends on number of environmental and laboratory factors including assay techniques and kits used, apart from inherent internal physiological factors.
Kanai and Shimizu (1984) found decreased basal LH levels towards the mid luteal phase and then progressively increased during the follicular phase. The same trend was noticed in present study, where LH levels were found lower on day 7 and 10 and increased on day 26 and 27 of protocol. Inadequate LH pulse frequency and low concentration of insulin impede the follicular growth and reduces the chance of ovulation. These conditions appear to occur in a state of under-nutrition/malnutrition (Roche, 2006) or lactation stress as was observed in buffaloes of Group III in present study. In a state of negative energy balance, endogenous opioids increase which in turn decrease the pulsatile secretion of LH. In addition to these, prolactin, oxytocin, cortisol, ACTH secreted in response to lactation, suckling and stress also reduce the LH pulse frequency and subsequently impair the follicular growth. Ultimately, follicles become atretic and then regress. The process of follicular growth and regression occur over and again till the above affairs persist resulting into anovulatory anestrus
(Kumar et al., 2014). Excessive clearance of ovarian steroids also leads to anovulation
(Walsh et al., 2007) and delayed luteal regression
(Petersson et al., 2006). The LH output is suppressed during the entire period of anestrus, however, rhythmic FSH secretion may initiate the emergence of follicular development up to preovulatory size. Apparently, the absence of an LH drive prevents ovulation of large follicle in anestrus buffalo, however, ovarian antral follicular turnover is not impaired during anestrus, which closely resembles to that of cycling buffaloes
(Sarath et al., 2016). Reduced plasma glucose concentration mainly affects the LH concentration and its modulation within the CNS at specific receptor, since glucose has specific effects on ovarian cells, including synergism with gonadotrophins. Adequate LH pulse frequency is required for growth and development of follicle after emergence of follicular wave.
Senatore et al., (1996) found a negative effect of energy deficit on ovulation during early lactation in first lactation dairy cows. Energy restriction impairs pulsatile LH secretion by decreasing the hypothalamic release of LHRH. The relatively poor response to synchronization treatment found in postpartum acyclic buffaloes as compared to post-pubertal acyclic heifers could be therefore justified on above ground.
The plasma estrogen profile revealed non-significant differences between periods in double Ovsynch treatment groups (Table 6) and varied from 6.95±2.36 to 14.06±7.42 ng/ml across different periods. The values were apparently higher in heifers than postpartum buffaloes. However, the plasma estrogen level did not correspond with the ovarian follicular dynamics recorded in estrus synchronized animals, wherein a clear period effect of treatment was noted. This may be due to failure of assay kits used to detect actual hormone or some other unknown reasons. The values of estradiol reported by
Varughese et al., (2014) and
Jerome et al., (2016) were lower than the present findings. However, an unique peak and basal profile expected over a normal estrous cycle was not observed in the present study due to multiple injection of GnRH and PG at various intervals. In present study, increased levels of estrogen, though non-significant, were noticed on day 26 and 27 of protocol.
Sarath et al., (2016) found significantly (
p<0.01) higher mean serum estradiol-17β concentration on day 0 than the other days of normal cycle and opined that this may be due to presence of dominant follicle secreting higher amount of estrogen during estrus. They further reported a smaller peak of estrogen during mid-luteal phase (day 10) indicating the presence of wave like follicular development and dominant follicle secreting higher estrogen.
The mean plasma progesterone concentrations were low towards basal values (<0.5 ng/ml) on the day of initiation of treatment in acyclic buffaloes of all three groups. The levels increased significantly (
p<0.001) on day 7,
i.e. just before PGF
2α injection in group II and III; on day 10 of protocol the levels dropped to the basal value in group I, but it was >0.5 ng/ml in group II and III; on the day 24,
i.e. just before 2
nd PGF
2α injection, significant rise in the level of progesterone noticed in all the groups, which dropped within 48-72 hr due to complete luteolysis to the basal values as observed on days 26 and 27 in all three groups coincidental to induced estrus, when FTAIs were performed (Table 5). These levels again increased significantly (
p<0.001) on day 12 post-AI in all three groups and on day 21 and day 35 post-AI in groups I and II, due to estruses being ovulatory with development and maintenance of CL and establishment of pregnancy in some of these animals.
In the present study, we found significantly increased levels of plasma progesterone in all animals on day 24 of protocol (before 2
nd PGF
2α injection). Other recent studies have also suggested that greater circulating concentrations of progesterone at the time of PGF
2α can improve fertility
(Martins et al., 2011). Greater progesterone concentrations during growth of the dominant follicle in a double Ovsynch protocol resulted in smaller ovulatory follicle size
(Wiltbank et al., 2011). Martins et al., (2011) recently reported greater rates of luteolysis for primiparous cows compared with multiparous cows treated with Ovsynch-56. Previous studies have shown that incomplete luteal regression resulting in elevated progesterone concentrations of > 0.50 ng/mL
(Souza et al., 2007) near TAI reduced P/AI by more than 50% in animals treated with Ovsynch. Thus, inadequate circulating progesterone in multiparous cows, coupled with potentially lower rates of luteal regression may partially explain the reduced effectiveness of double ovsynch in multiparous cows. We found increased circulating progesterone concentration on day 12 post-AI in all groups. It was also clearly observed that buffalo with low progesterone at day 21 and 35 post-AI had substantially reduced fertility and corroborated with the observations of
Herlihy et al., (2012).
The plasma levels of insulin in Jaffarabadi animals under study varied from 64.37±5.45 to 157.46±3.62 pg/ml on different periods of treatment across groups. There were no significant differences between periods in the plasma insulin levels in animals of any of the treatment groups. However, the mean values on all days for postpartum acyclic buffaloes of group-III were significantly (
p<0.01) lower than those of post-pubertal acyclic heifers in group-I and group-II (Table 6). These findings concurred well with the previous report of Sheetal (2017) in cows. Growth and maturation of follicle depends upon bioavailability of insulin. Low insulin concentration might limit the responsiveness of ovary to endogenous gonadotropin secretion thus affecting ovulation (Butler and Smith, 1989). Insulin stimulates the release of GnRH from hypothalamus and LH from pituitary
(Tanaka et al., 2000). Insulin regulates CL function by increasing glucose availability and thus production of hormone in cow
(Sousa et al., 2016). In addition, insulin may also act on the pituitary gland to increase gonadotroph sensitivity to GnRH
(Solorzano et al., 2010).
Blood biochemical profile
The blood glucose levels varied from 63.20±4.51 to 79.00±1.87 mg/dl across different periods of treatment and groups. The values of blood glucose were higher at most of the intervals in group-I heifers followed by Group-III buffaloes and Group-II heifers with significant (p<0.001) differences on day 17. There were no significant differences in the blood glucose levels between days/periods in any of the groups (Table 6).
Sharma et al., (1998) reported that the hypoglycemic state in buffaloes reduced the hypothalamic-hypophyseal-ovarian axis signal transmission leading to anestrus condition.
Jayachandran et al., (2013) revealed no significant variation in plasma glucose level between anestrus and regular cyclic buffaloes.
The levels of plasma total cholesterol in animals under study varied from 57.40±5.44 to 96.60±7.55 mg/dl across different periods of treatment and groups without significant differences (Table 7). The present value of plasma total cholesterol was in accordance with the report of
Sharma et al., (1998) and
Prajapati et al., (2018), but almost one half to those reported by
Singh et al., (2004), Parmar et al., (2012) and
Selvaraju et al., (2017). Henricks et al., (1971) were of the opinion that the highest adrenal cholesterol concentration occurs at estrus, when the females are under estrogen dominance eventually facing a decline later, when the progesterone phase sets in.
The plasma total protein level varied from 5.90±0.22 to 7.14±0.17 g/dl irrespective of different periods of treatment and groups. The mean values of plasma total protein increased with advancing age and differed significantly (
p<0.001) at most of the periods, the values were lower in group I heifers and higher in postpartum buffaloes of group III (Table 7). These findings with respect to values coincided well with the previous reports of
Singh et al., (2004), Jerome et al., (2016) and
Prajapati et al., (2018). However, other reserchers
(Parmar et al., 2012; Chaudhary et al., 2018) reported higher values of total protein than the present findings. Optimum protein level is necessary for the development of endocrine and sex organs. Srivastava and Kadu (1995) reported significantly higher levels of serum total protein in cycling heifers as compared to delayed pubertal heifers (7.54±0.11 vs 6.64±0.66 g/dl).
