The present study was planned to assess the oestrus induction and fertility response in anoestrus Frieswal cross-bred heifers subjected to various treatment protocols as; G1 (n=12) - Intra-vaginal progesterone implant protocol; G2 (n=13) Inj. phosphorus and vitamins A, D, E supplements G3 (n=12) - Inj. GnRH followed by PGF2α, G4 (n=12) - Tab. Clomiphene citrate followed by Inj. GnRH, G5 (n=12) - Oral Levonorgestrel and Ethinylestradiol. Highest oestrus induction response and conception rate was recorded in G1 as 100% and 68.75% respectively, as compared to others. The duration for oestrus exhibition was significantly lower in groups G1, G3 and G4 as compared to others. The blood plasma levels of calcium, phosphorus, copper and manganese were significantly low in anoestrus Frieswal heifers as compared to normal cyclic heifers. Whereas, the levels of cholesterol and zinc did not differed significantly between both the groups. Better fertility response was observed using hormone based oestrus induction protocols.
The onset of recurring or cyclic periods of sexual receptivity when female becomes sexually mature and able to reproduce is referred to as puberty. It is also the age at first ovulation and is characterised by an increase in plasma progesterone concentrations above 1 ng/ml (Evans and Rawlings, 1995). To achieve higher reproductive efficiency in one’s lifetime, the onset of puberty at optimum time is the prerequisite. Delayed puberty is one of the most frustrating problems associated with sub-fertility in crossbred dairy heifers in India. The condition usually occurs due to one or more factors like hormonal imbalance, poor body condition score and deficiency of certain minerals and vitamins etc which leads to heavy economic losses to the dairy farmers in terms of less milk yield and calf crop produced by a cow in her lifetime. It was observed that, even after attainment of pubertal age and body weight a large proportion of dairy heifers fail to commence cyclicity (Singh et al., 1998). This necessitates use of exogenous therapeutics to induce oestrus and improve fertility in heifers. Several therapeutics are available and have been tried with variable degree of success like herbal (Markandeya et al., 2002), hormonal (Khasatiya et al., 2004), vitamin and mineral supplements (Singh et al., 2006).
Therefore, the present study was planned to assess the oestrus induction and fertility response in delayed pubertal anoestrus Frieswal (Holstein Friesian x Sahiwal) cross-bred heifers subjected to various treatment protocols.
Materials and Methods
The present study was undertaken on sixty eight delayed pubertal Frieswal anoestrus cross-bred heifers of 24.13±0.13 month age and 267.38±2.67 kg body weight maintained under similar feeding and management conditions at Military Dairy Farm, Meerut (28°59’40″N, 77°43’58″E). Animals were selected after per rectal examination of reproductive organs twice at ten days interval and there was no genital abnormality except ovaries were smooth and inactive. The anoestrus animals were divided into five treatments and one control group. Twelve normal cyclic heifers were also followed under the study-
Group 1 (n=12)
An intra-vaginal progesterone implant (TRIU-B®) for 7 days followed by its removal and PGF2α (Inj. Lutalyse®) 2.5ml intramuscularly (I/M) on 8th day thereafter on 9th day Inj. estradiol benzoate @ 0.75 mg/animal I/M.
Phosphorus supplement (Inj. Tonophophan®)10ml I/M along with Inj. Vitamins A, D, E supplement (VETADE®) 10ml I/M on alternate days for 5 occasions.
GnRH analogue (Inj. Receptal®) 5ml I/M followed by PGF2α (Inj. Lutalyse®) 2.5 ml I/M on 8th day.
Tab. Clomiphene Citrate @ 300 mg/animal/day orally for 7 days followed by GnRH analogue (Inj. Receptal®) 2.5 ml I/M on 8th day.
Oral feeding of combination of 1.2 mg Levonorgestrel (progesterone) and 0.24 mg Ethinylestradiol (oestrogen) along with mineral mixture @ 40g/day/animal for 10 days.
Control, received no treatment.
Oestrus detection was carried out twice daily during the cooler hours (6 AM and 5 PM) of the day by visual observations and confirmed by presence of tonicity and turgidity in the reproductive tract during per-rectal examination. The animals detected in oestrus were inseminated with frozen thawed semen twice during each oestrus at 10-12 h interval. Pregnancy was confirmed 60-75 days post-insemination by per-rectal examination. Before the start of the experiment, blood samples were collected from jugular vein using 16G sterile needles in heparinised tubes which were than centrifuged and plasma was stored at −20°C until mineral and biochemical estimations. Estimation of zinc, copper and manganese was done by Atomic Absorption Spectroscopy after wet digestion in tri-acid (Raghuramulu et al., 1983). Calcium, phosphorus and total cholesterol estimations in blood plasma were done as per the protocols in the kits (Transasia®). Data were analysed using standard statistical methods (Snedecor and Cochran, 1994).
Results and Discussion
The oestrus induction and fertility response using various treatments has been worked out (Table 1).
Table 1: Oestrus induction and fertility response in anoestrus Frieswal heifers
|Groups||Oestrus response (%)||Time taken for oestrus exhibition from the start of treatment (Mean±SEM in days)||Conception rate (%)||Av. service per conception|
|Untreated anoestrus control (n=7)||28.57(2/7)||–||28.57||3.50|
|Normal cyclic control (n=12)||100 (12/12)||–||70.58||1.41|
Values bearing different superscripts differs significantly (P < 0.05)
The oestrus induction response recorded under present study was lowest in untreated anoestrus control group and highest in G1, similar to normal cyclic control animals. The conception rate was highest in G1 as compared to other treatment groups and untreated control group. The duration for oestrus exhibition from the start of treatments was significantly (P < 0.05) lower in groups G1, G3 and G4 as compared to others. The overall fertility response under present findings was best for G1 as compared to other treatment groups.
In agreement to our study, Buhecha et al. (2015) also found 100% oestrus induction response with 50% conception rate in anoestrus crossbred cows using TRIU-B intravaginal protocol, however we observed higher conception rate. Similarly, other researchers have also reported 100% oestrus induction response using different hormonal protocols (Markendeya and Bharkad, 2004, Chaudhari et al., 2012, Dhami et al., 2015). Mathur et al. (2011) observed 70-80% oestrus induction response in anoestrus Frieswal females following oral administration of Levonorgestrel and Ethinylestradiol with mean duration for exhibiting oestrus ranging from 14.7±4.99 to 27.7±3.76 days, which corroborates to our findings. Progesterone may be used to induce oestrus in animals (Willemse et al., 1982). However, the combination of progesterone and oestrogen was effective therapy to induce oestrus (Mathur et al., 2004) in buffalo heifers (Shanker et al., 1996), cattle heifers (Thakur and Bhatt, 1999) and postpartum cows (Kumar et al., 2000). Mathur et al. (2005) observed that 80% Frieswal and 66.6% Sahiwal heifers expressed oestrus activity with 100% fertility after administering vitamin A and Tonophosphan, however we observed lesser response in Frieswal heifers under present findings. Similarly, Markendeya et al. (2002) documented 75% oestrus response with 1.9 services per conception after micro mineral and vitamin E supplementation in anoestrus Deoni cows. In a study by Parmar et al. (2012), the oestrus induction response in postpartum anoestrus buffaloes treated with Inj. Receptal® and Tono-Vitacept was 83.33% and 50.00, respectively. The corresponding figures for oestrus induction interval were 20.90 ± 3.13 and 27.14 ± 3.74 days and the overall conception rate was 80% and 71.42 %, respectively. Up to 80% oestrus induction response was observed in anoestrus crossbred heifers after feeding vitamin-mineral supplements (Akhtar et al., 2004). Srivastava (2008) reported 93.33% oestrus induction response in anoestrus crossbred heifers following mineral mixture supplementation for 20 days with 28.36 days average oestrus induction interval from initiation of treatment. These findings are in line with our present study.
Dugwekar et al. (1980) found 97.5% oestrus induction response with 57.1% anoestrus cows conceived after receiving Clomiphene citrate mixture. Similarly, in agreement to our studies 66.7% of pre-pubertal heifers had ovulations following treatment with Clomiphene citrate (Rehman et al., 2014). Clomiphene citrate is largely regarded as anti-oestrogen, it was expected to remove the negative feedback effect of estrogen on the release of gonadotropins, resulting in an increased release of gonadotropins, which would support follicular growth and further administration of GnRH analogue may support ovulation.
Table 2: Blood plasma mineral & biochemical parameters of Frieswal heifers at Military Farm, Meerut
|S. No.||Attribute||Anestrus (N=68)||Normal cyclic (N=12)|
|1||Calcium (mg/dL)||8.46 ± 0.16a||9.75 ± 0.36b|
|2||Phosphorus (mg/dL)||3.88 ± 0.13a||5.63 ± 0.26b|
|3||Cholesterol (mg/dL)||91.98 ± 3.05||96.43 ± 5.88|
|4||Zinc (µg/mL)||0.94 ± 0.05||1.14 ± 0.14|
|5||Copper (µg/mL)||0.38 ± 0.03a||0.96 ± 0.08b|
|6||Manganese (µg/mL)||0.26 ± 0.01a||0.38 ± 0.03b|
Values with different superscripts differ significantly (P < 0.05) between groups
The blood plasma levels of calcium, phosphorus, copper and manganese were significantly (P < 0.05) low in anoestrus Frieswal heifers as compared to normal cyclic heifers. Whereas, the levels of cholesterol and zinc did not differed significantly between both the groups under present study. Lower serum calcium levels were observed in delayed pubertal anoestrus heifers than normal cyclic heifers by several researchers (Kalita et al., 1999; Singh et al., 2005). Similarly, lower blood serum or plasma calcium and phosphorus levels were opined by several authors; in acyclic buffaloes as compared to cyclic animals (Dhoble and Gupta, 1986; Virmani et al., 2011), infertile Surti buffaloes than fertile buffaloes (Shah et al., 2003), non-cyclic anoestrus as compared to cyclic crossbred cows (Yadav et al., 2004). Recently, Singaram and Jacob (2017) also documented significantly low level of inorganic phosphorus in anoestrus in comparison to regular cyclic animals. Anoestrus animals had lower serum phosphorus, zinc and copper levels than the control (Ceylan et al., 2008). Likewise, lower serum copper levels were documented in anoestrus as compared to cyclic animals (Dutta et al., 2001). Similar trend in buffalo was reported by Singh et al. (2006). All these studies are in agreement to our results.
Reproductive failure accounts for more than half of all losses resulting from disease mostly due to mineral deficiency (Kaikini, 1989). Calcium and Copper plays important role in the process of steroidogenesis in ovaries (Velduis et al., 1982), Copper deficiency may also be considered responsible for the anoestrus conditions as this is a component of enzymes involved in steroidogenesis (Hidiroglou, 1979). Deficiency of phosphorus is responsible for irregular oestrus, anoestrus, decreased ovarian activity and conception rate (Pugh et al., 1985). Improper calcium and phosphorus ratio is also related with GnRH and LH release from pituitary cells (Naor and Catt, 1981).
It is indicated from the present study that progesterone and oestrogen hormone based oestrus induction protocols can be the best resort to overcome the problem of anoestrus due to delayed puberty in dairy heifers, subjected to the condition that all the nutrients specially the mineral and vitamins supplements may be adequately incorporated in the ration.
We thank Director, ICAR-CIRC, OIC and Veterinary Officer Military farm, Meerut, Dr. A.K. Mathur (Ex-Principal Scientist) and all the section In-charges for their constant help support during this study.