The blood plasma progesterone concentrations were estimated by radioimmunoassay precoated tube method with the help of RIA kits supplied by FAO/IAEA Agricultural and Biotechnology, Seibersdorf, Austria. The mean intra- and inter-assay coefficients of variation were 6.71 and 10.08 % respectively. The sensitivity of the assay was 0.032 ng/ml.

The various abnormalities identified and their incidences in repeat breeder cattle and buffaloes have been depicted in Table 1. Out of 183 repeat breeding cattle, more number of cattle (31.70%) were suffered due to a combination of more than one factors i.e. multiple factor (Gp V), followed by hormonal aberrations (Gp III; 28.42 %), genital tract infections (Gp II; 18.03 %), poor management (Gp IV; 14.75 %) and acquired anatomical abnormalities (Gp I; 7.10 %). The number of repeat breeding buffaloes in respective categories were 8 (25.80 %), 8 (25.80 %), 8 (25.80 %), 5 (16.14 %) and 2 (6.46 %).

Figure 1

Table 1: Incidences of factors causing repeat breeding syndrome in cattle and buffaloes.

Out of the animals belonging to Gp I, 8/13 (61.54 %) cattle and 2/2 (100 %) buffaloes were repeater due to hard and kinked cervix. In the present study the incidence of OBA was found 2.2 % (4/183) in cattle whereas, no buffalo had OBA which might be due to less number (n=31) of buffaloes presented at field during the study. Among Gp I animals one cow and no buffalo were found with uterine tumor. The plasma P₄ profiles of Gp I animals were comparable with that of normal animals (Table 2).

Out of Gp II animals most of the cattle (26/33) and all the buffaloes were suffering from endometritis. The incidence of salpingitis in the present study was 2.7 % (5/183) in cattle whereas, the condition was not encountered in buffaloes. However, two cattle had combination of endometritis and salpingitis. The plasma P₄ profiles of Gp II animals were comparable with that of normal animals (Table 2).

Figure 2

Table 2 : Mean (±S.E.) plasma progesterone profiles of normal and repeat breeder cattle and buffaloes.

The plasma P₄ profiles of Gp III animals have been depicted in Table 3. Among this group, maximum numbers of cattle were having cystic ovarian degeneration (19/52), followed by delayed ovulation (17/52) and luteal insufficiency (16/52), whereas, buffaloes in this group had delayed ovulation (50%) and early luteal deficiency (50 %). Among the delayed ovulators, 10 cows and 4 buffaloes had suprabasal P₄ (0.87±0.21ng/ml & 0.50±0.07 ng/ml respectively) on the day of estrus. All these animals had prolonged estrus period (3-5 days). However, 7 cattle showed prolonged estrus even with basal progesterone level (0.14 ± 0.02ng/ml) comparable to that of normal animals on the day of estrus.

Out of the cattle suffering from luteal insufficiency, maximum number of cows were early luteal deficient (day 5 post-ovulation 68.7 %), followed by mid (days 5-10 post-ovulation; 12.5%), late (days 10-15 post-ovulation; 12.5 %) and during both early and mid (up to day 10 post-ovulation; 6.25 %) luteal deficient.

Figure 3

Table 3: Mean (± S.E.) progesterone profiles (ng/ml) of repeat breeding cattle and buffalo having progesterone aberrations on different days of estrous cycle.

Despite having clear cervicovaginal mucus, anatomically normal genitalia and normal plasma P₄ profiles (Table2), 14.7 % (27/183) cattle and 16.1% (5/31) buffaloes of Gp IV were repeater due to managemental and nutritional factors (Table 1). Either they had poor BCS (1.5-2.0) or inseminated with poor quality semen. Poor semen quality or faulty AI techniques were found to be major managemental factors in cattle (25/27) and buffaloes (2/2).

In the category of Gp V animals (Table 4), the combination of genital infections and hormonal aberrations was the major contributing factor to repeat breeding (Cattle- 23/58; Buffaloes- 3/8), followed by combination of anatomical defects and hormonal aberrations (Cattle-19/58; buffaloes- 1/58); genital infection and anatomical defects (Cattle- 19/58; Buffaloes- 1/58).

Figure 4

Table 4: Mean (± S.E.) progesterone profiles (ng/ml) of repeat breeding cattle and buffalo with multiple etiology on different days of estrous cycle.

Higher incidence of repeat breeding due to combination of multiple etiologies than other single causes was observed in the present study. This is the fact that lack of expertise and inappropriate interventions at farmer’s doorsteps exaggerate the condition of repeat breeding syndrome and hence involve multiple factors to complicate the situation. Rao (1981) also observed relatively higher incidence of functional abnormalities (73.14 %) followed by infections (20.85 %) and anatomical abnormalities (6.0%) in crossbred cows.

Many of known inherited causes of infertility involve anatomical abnormality like kinked cervix. The hardness of cervix may appear due to trauma or lacerations during calving and AI followed by infection and fibrosis. The incidence of cervicitis varied from 0.9 – 5.52% in cattle (Mylrea, 1962; Rao, 1981) and 0.5 – 2 % in Indian buffalo (Kumar et al., 1988). The another important anatomical anomaly i.e. ovarobursal adhesions (OBA) develop between ovary and ovarian bursa as a result of faulty handling of ovary, intrauterine infusion of irritating drugs in large volume under pressure and due to infections. The incidence of OBA has been reported earlier as 0.29 - 1.8 % in cattle (Rao, 1981; Dobson and Kamonpatana, 1986) and 0.8-2.0 % in buffaloes (Rao and Sremannarayana, 1982). However, in the present study the incidence of OBA was found 2.2 % (4/183) in cattle whereas, no buffalo had OBA which might be due to less number (n=31) of buffaloes presented at field during the study. Tumors of bovine reproductive organs are quite rare (Salisburry, 1985) while the incidence of uterine tumor in buffaloes has been reported very low i.e. 0.3 – 0.72 % (Agarwal and Tomer, 1998).

The endometritis and pyometra are the most commonly encountered anomalies causing infertility in cattle and buffaloes under field or farm conditions. The incidence of endometritis has been reported to be 3.0 – 13.97 % in cattle (Zemjanis et al.,1961; Rao, 1981) and 2.4 – 20.0 % in buffaloes (Rao and Sreemanarayana, 1982) during clinical investigations. Slaughterhouse studies and clinical investigations revealed higher incidence of endometritis in buffaloes than in cattle (Dobson and Kamonpatana, 1986). The reason being habit of wallowing, not closely opposed vulval lips and improper postpartum care renders buffaloes more prone to endometritis (Agarwal and Tomer, 1998).

Salpingitis is one of the major disorders of fallopian tube. The incidence varied from 2.2 –3.6 % in cattle (Mylrea, 1962; Rao, 1981) and 0.8 – 4.5 % in buffaloes (Agarwal and Tomer, 1998).

The cystic ovarian degeneration was not encountered in buffaloes in the present study. The incidence of cystic ovarian degeneration observed in earlier studies varied from 8.8 – 27.4 in cattle (Tanabe and Brofee, 1982; Zemjanis, 1987) and 0.9 – 2.0 % in buffaloes

(Rao and Sreemanrayana, 1982; Kumar and Agarwal, 1986). The lower incidence of cystic ovarian degeneration in buffaloes may be due to lower stress of milk production than dairy cows (Agarwal and Tomer, 1998). The plasma P₄ concentration more than 0.35 ng/ml at the time of standing estrus is considered as suprabasal progesterone, which may be a cause of prolonged estrus (Bage et al., 2002). The fate of dominant follicle is dependent on Luteinizing hormone (LH). The LH secretion is governed by combination of P₄ and estradiol, as higher P₄ inhibit the LH pulse frequency, whereas, the higher estradiol inhibit the LH pulse amplitude (Goodman and Karsch, 1980). Hence, the suprabasal P₄ causes decrease in LH pulse frequency thereby responsible for dominant follicle to grow up to a larger size and stay for longer duration and secretion of higher estradiol concentarion (Bigelow and Fortune, 1998) and consequently prolonged exhibition of estrus. Various other workers (Kimura et al., 1987; Kasrija et al., 2006) also observed higher incidence of progesterone deficiency during early phase of estrous cycle. Normal early embryonic development and pregnancy were found to be associated with P₄ activity initiated on days 4.5 – 10 postinsemination (Laming et al.,1989; Alhibin et al., 1991; Larson et al., 1997). Delayed function of CL either alone or in combination with lowered secretion of P₄ during luteal phase is one of the major causes of repeat breeding syndrome (Thatcher et al., 1994). There could be possibility that luteal inadequacy due to diminished response to circulating LH may account for early embryonic mortality leading to repeat breeding syndrome (Shelton et al.,1990).

Besides, genetic, infectious and endocrine factors, managemental and nutritional factors also play an important role in causation of repeat breeding syndrome. The BCS is a useful field tool for estimating changes in body weight, tissue reserve and for assessing dairy managemental practices and ration. The recommended BCS for a dairy cow is 3.25-4.0 (Edmondson et al., 1989). Large negative energy balance during post partum period is associated with reduced BCS and subfertility (Royal et al., 2000).

In the present study, there was a slack in any one of the steps involving storage, thawing, post-thaw handling of semen, incorrect and inappropriate insemination with respect to stage of estrus. Correctness and precision of these steps are mandatory in achieving high success rate with AI (Salisburry et al., 1985).

Involvement of more than one factor for causing repeat breeding was observed in maximum number of cattle and buffaloes in the present study. Kutty and Ramchandran (2001) also observed high incidence of repeat breeding because of combination of ovulatory disturbances and reproductive tract infection. The results of other combination factors could not be compared due to scarcity of similar type of work.

Hence, the present study indicated that hormonal aberrations and its combination with other factors constitute the major cause of repeat breeding syndrome in dairy animals.

The authors are thankful to IAEA (International Atomic Energy Agency), Austria for providing progesterone kits for this research work.