Introduction
Fixed timed artificial insemination (FTAI) is nowadays very popular, and it has cost-effective reproductive technology, which increases the overall genetic merit of a herd as well as increases the profitability of a farm [1]. By implementing the FTAI programs in a herd’ more numbers of cows can be impregnated at a predetermined time with a better conception rate [1]. The key is to select a protocol that fits your needs regarding cost, labor, time, animal handling, experience, and other factors. Mizoram has the most difficult terrain; the topography being by and large mountainous with precipitous slopes forming deep gorges and culminating into several streams and rivers. In a mountainous region like Mizoram, the availability of the drugs, repeated administration of the drug and repeated examination of animals are quite challenging [2]. Recently, G6G, the pre-synchronization protocol was applied in a huge number of animals, resulting in better pregnancy rates but this method has disadvantages about the period, labor, and cost of each medicine [1]. Anestrus and infertility during the postpartum period of dairy cattle were documented as major problems in a dairy herd [3]. Estrus synchronization research is imperative to establish optimal doses and agents to use for favorable synchrony and fertility in each breed and introduce a simple, pragmatic, and reliable protocol that can be adopted by the farmers for better conception. The present study aimed to evaluate the reproductive performance of post-partum anestrus cows synchronized with select-synch and modified select-synch programs (Seld-synch) to enhance the conception rate.
Materials and Methods
The Institution Animal Ethics Committee duly approved the experimental design of the study. The study was conducted on 164 crossbred cows at 2nd to 5th lactation in and around the Aizawl district of Mizoram, India. The experimental animals were dewormed before treatment and examined per rectum to study their ovarian and genital status. All cows were kept in an intensive rearing system in which they received a TMR formulated (CP-20% and TDN-75%) for lactating dairy cows producing 15 kg of 3.5% fat milk and had free access to drinking water. Total mixed rations were fed twice daily (morning and evening) along with available green grasses and tree leaves. All the cows participating in this experiment were milked twice daily at approximately 8-hour intervals and monitored daily for signs of disease. If any health-related issues occurred, animals were moved to isolated pens appropriate treatments were performed and the respective cows were removed from the rest of the study. The experimental cows were divided into two groups comprising 82 animals in each group. In group A, the modified select-synch protocol (Seld-synch) was applied and in group B, select-synch protocol was applied. The pregnancy was confirmed by per rectal examination after 90 days of gestation.
Ten ml of blood samples were collected on day 0 (before starting the protocols) and day 1 (before AI) in a vacuum clot activator vial containing no additives by jugular puncture with a sterile 18 gauge needle fitted with a plastic syringe. Following standing at room temperature for 20 minutes, the clot activator vials were centrifuged at 3,000 rpm for 10 minutes. Then, the obtained sera were kept at −20°C until analysis. The serum levels of glucose, total cholesterol, calcium, and phosphorus were analyzed with the help of the FUJIFILM (DRI CHEM 4000i) autoanalyzer, and the blood hormonal profile was analyzed by a commercial ELISA kit.
The estrous cycle of postpartum anoestrus cow in group A was induced by injection of synthetic prostaglandin analog, i.e., cloprostenol sodium (PGF2α) at 500 µg through the intramuscular route (i/m) at day 0 (zero) followed by buserelin acetate injection at 20 µg along with vitamin A injection 10 ml i/m at day 3, PGF2α at 500 µg i/m at day 11. The time of AI was fixed at 48–72 hours of second post PGF2α injection. At the time of FTAI, another buserelin acetate injection at 20 µg was injected through the intramuscular route. The post-AI progesterone injection was given to all the treated animals at 500 mg on days 10 and 17 through the intramuscular route.
In group B animals, estrus was induced by an injection of synthetic prostaglandin analog, i.e., cloprostenol sodium (PGF2α) at 500µg i/m at day 0 (zero) followed by buserelin acetate injection at 10 µg i/m at day 2, PGF2α at 500 µg i/m at day 8. At the time of FTAI, another buserelin acetate injection at 10 µg was injected through the intramuscular route. The time of AI was fixed at 48–72 hours of second post PGF2α injection [4].
The data obtained from the study were subjected to statistical analysis using a suitable formula for meaningful and accurate comparison and interpretation as per Snedecor and Cochran [5]. Mean values of the blood biochemical and hormonal profiles were compared by “t-test” and the other parameters were compared by chi-square test. The significance of differences was interpreted by Duncan’s Multiple Range Test using the SPSS 20.0 version.
Results
In the present study, it was observed that the majority of post-partum anoestrus cows (86.66%) exhibit oestrus in the treatment group A in comparison to the group B (13.34%). The chi-square test revealed that the values were significantly differed (p < 0.01) (Table 1). The oestrus signs, i.e., bellowing, restless, standing to be mounted, mounting over the other animals, swollen and edematous vulvar lips, copious, clear, stringy mucous discharge from the vagina, moist and congested vulvar mucus membrane were pronounced in the majority of the cattle in the treatment group A (92.68 %). The Chi-square test revealed that the values were significantly differed (p < 0.01) (Table 1). The cervix was softer and remained open in the majority of post-partum anoestrus cows (93.33%) on the day of fixed-timed AI in group A and the values significantly differed (p < 0.01) (Table 1). The good uterine tone was observed in the majority of post-partum anoestrus cows (81.00%) on the day of fixed-timed AI and the values significantly differed (p < 0.01). Most of the post-partum anoestrus cow (93.33%) ovulates in a normal manner after the applying modified select-synch protocol (Seld-synch). The chi-square test revealed that the values significantly differed (p < 0.01) between the treatment groups (Table 1). In the present invention, it was observed that the majority of post-partum anoestrus cows of group A exhibit oestrus signs before 48 hours of the second post PGF2α injection (60.97%) in comparison to 48 hours (17.07%) and 72 hours (21.95%) and the majority of cattle were inseminated at 48 hours (78.00%). The Chi-square test revealed that the values were significantly differed (p < 0.01) (Table 2).
Table 1.
Efficacy of select-synch versus modified select-synch protocol for the therapeutic management of post-partum anestrus in dairy cow (n=164).
| Sl No. | Parameter | Group A | Group B | p-value | X2 value |
|---|---|---|---|---|---|
| 1 | Exhibit oestrus (%) | 86.66 | 13.34 | 0.00 | 107.54** |
| 2 | Signs of oestrus: bellowing, restless, mounting, swollen vulva, copious, clear, stringy vaginal mucous discharge, congestion of vulval mucous membrane (%) on the day before FTAI | 92.68 | 7.32 | 0.00 | 145.75** |
| 3 | Cervix: Softer and remains open on the day of FTAI (%) | 93.33 | 6.67 | 0.00 | 150.22** |
| 4 | Ovulation on the day of Fixed-Timed AI (%) | 93.33 | 6.67 | 0.00 | 150.22** |
| 5 | Good uterine tone (%) | 81.00 | 15.00 | 0.00 | 149.25** |
| 6 | Conception rate (%) | 87.81 | 12.19 | 0.00 | 114.39** |
**p < 0.01.
Table 2.
Time of insemination of post-partum anoestrus cow after post-therapy (n=164).
| Parameter | 48 hours | 72 hours | p-value | X2 value |
|---|---|---|---|---|
| Cattle (%) | 78.00 | 22.00 | 0.00 | 62.74** |
**p < 0.01.
There were significant differences in blood glucose, cholesterol, calcium, phosphorus, estrogen, and progesterone levels between day 0 and day 1 (p < 0.01) within the treatment group (Table 3). In the present study, there were significant differences (p < 0.05) in cholesterol values within the groups, i.e., day 0 and day 1. At day 0, it ranged from 130.00 mg/dl to 133.40 mg/dl. The level of serum cholesterol on day 1 (after treatment) ranged from 159.10 mg/dl to 166.40 mg/dl which was higher than that of day 0. There was a significant difference in calcium and phosphorus levels between day 0 and day 1 (p < 0.01) within the group where the levels of calcium and phosphorus were higher on day 1. The level of serum estrogen of postpartum anestrus cow (at day 0) was 4.50 pg/ml, on the other hand, at day 1(after therapy), it ranged from 6.91 to 7.69 pg/ml. There were significant differences in progesterone values between day 0 and day 1 (p < 0.01) within the groups.
There was much improvement in conception rate (87.81%) in the treatment group A in comparison to the group B (12.19%). The chi-square test revealed that the values were significantly differed (p < 0.01) (Table 1).
Table 3.
Level of blood biochemical and hormonal profiles in post-partum anestrus cow at day 0 (before treatment) and day 1 (day before FTAI) (mean ± SE ) (n=10).
| Sl. No | Parameters | Days | Group A | Group B | t-value |
|---|---|---|---|---|---|
| 1 | Glucose (mg/dl) | 0 | 41.10 ± 3.79 | 38.20 ± 4.65 | 0.483NS |
| 1 | 71.90 ± 4.43 | 71.80 ± 5.41 | 0.014NS | ||
| t-value | 5.272** | 4.704** | |||
| 2 | Cholesterol (mg/dl) | 0 | 133.40 ± 18.08 | 130.00 ± 10.24 | 0.164NS |
| 1 | 166.40 ± 14.68 | 159.10 ± 17.87 | 0.316NS | ||
| t-value | 1.416* | 1.413* | |||
| 3 | Calcium (mg/dl) | 0 | 7.74 ± 0.38 | 7.41 ± 0.32 | 0.657NS |
| 1 | 9.75 ± 0.43 | 9.58 ± 0.50 | 0.241NS | ||
| t-value | 3.193** | 3.885** | |||
| 4 | Phosphorus (mg/dl) | 0 | 5.90 ± 0.45 | 6.27 ± 0.38 | 0.619* |
| 1 | 7.59 ± 0.37 | 7.10 ± 0.54 | 0.746* | ||
| t-value | 2.030* | 1.977* | |||
| 5 | Estrogen (pg/ml) | 0 | 4.50 ± 0.67 | 4.50 ± 0.67 | 0.00NS |
| 1 | 7.69 ± 0.32 | 6.91 ± 0.29 | 0.295* | ||
| t-value | 4.242** | 4.507** | |||
| 6 | Progesterone (ng/ml) | 0 | 6.83 ± 0.93 | 6.78 ± 0.84 | 0.04NS |
| 1 | 1.22 ± 0.39 | 1.76 ± 0.17 | 1.276** | ||
| t-value | 4.992** | 6.440** |
**p < 0.01; *p < 0.05; NSNon significant.
Discussion
The early exhibition of oestrus, pronounced oestrus signs in group A animals could be due to the peak activity of estradiol in the blood circulation of group A animals (7.69 ± 0.32 pg/ml) in comparison to group B animals (6.91 ± 0.29 pg/ml) after post therapy which prepares for subsequent ovulation. Similar results were also reported by Malik et al. [6] who suggested that cows that exhibited estrus after estrus synchronization might have required concentrations of estradiol necessary to effectively prepare follicular cells for luteinization. Beneficial input of vitamin A towards the development of the oocyte as well as steroidogenesis for the maintenance of reproductive tissues, regulation of ovarian function, and ovulation is also one of the factors for maximum oestrus response, and ovulation in group A animals [7].
The timing of insemination is most important in FTAI as it can affect pregnancy rate which is associated with estrus, ovulation, and rates of fertilization [8]. It was observed that the majority of post-partum anoestrus cows of group A exhibited oestrus signs before 48 hours of the second post PGF2α injection and inseminated at 48 hours. It might be due to the action of estradiol which prepare for succeeding ovulation. Analogous results were also reported by Busch et al. [9] who suggested that cows that exhibited estrus after removal of CIDR may have attained concentrations of estradiol necessary to effectively prepare follicular cells for luteinization.
Glucose is considered the main energy substrate for the bovine ovary, and an adequate quantity of glucose is essential to maintain ovarian activity. Insufficient nutrient intake can reduce circulating glucose concentration [10]. In the present study, it has been observed that there was a significantly low level of blood glucose at day 0 (before treatment) in comparison to day 1 (after treatment) (p < 0.01) within the groups but between the groups the values were non-significant. The variation of the serum glucose level of postpartum anestrus cow might be due to inadequate availability of utilizable glucose and then there was a decrease in GnRH leading to a decrease in LH release, and thus delayed estrus response [1].
Cholesterol is a precursor for steroid hormones and plays an important role in the manufacture of bile acids, steroid hormones, and vitamin D [11]. The decrease in plasma cholesterol concentration resulted in the suppression of luteal function and eventually delayed ovulation in dairy cows [12]. The variation in the level of serum cholesterol might be attributed to several factors like the fertility status of the animal, the stage of the estrous cycle, and genotype [13].
There was a significant difference in calcium level between day 0 and day 1 (p < 0.01) within the group where the level of calcium was higher on day 1. The ratios of calcium and phosphorus between 1.5:1 and 2.5:1 for lactating cows should not affect reproduction. One of the main functions of calcium is to allow muscle contraction [11]. Uterine tonicity will also be adversely affected by the cow if circulating calcium is deficient and it may experience prolonged calving and retained placenta. In deficient cows, the uterine involution is not proper and it may impair post-partum fertility which leads to post-partum anoestrus [14].
There were significant differences in phosphorus values between day 0 and day 1 (p < 0.05) within and between the groups. In the present study, the lower level of serum phosphorus at day 0 in both groups might be associated with impairment in reproduction, decreased fertility rate, decreased ovarian activity, and ultimately post-partum anestrus in cattle [11]. After post therapy, the serum level of phosphorus was significantly increased (p < 0.05) in both the groups, and a higher level was recorded in group A animals which might be due to post-partum resumption of ovarian activity, folliculogenesis, and steroidogenesis after hormonal therapy [1].
In the present study, there were significant differences in estrogen values between day 0 and day 1 (p < 0.01) within the groups, on the other hand, there was a significant difference (p < 0.05) in estrogen level between the groups at day 1. In the present investigation, an increased level of estrogen on day 1, i.e., the day before AI might be the effect of buserelin acetate injection, and the response was higher in group A animals [1]. As buserelin stimulates the pituitary gland’s gonadotropin-releasing hormone receptor (GnRHR) which leads to a rise in FSH and LH levels, and that may be responsible for apposite follicular development, and the theca cells make more estrogen. As a result, estrogen concentration was higher which led to more LH receptors made by the theca cells, provoking theca cells to make more androgen that may make estrogen downstream. This regeneration loop causes LH to spike sharply, and it is this spike that causes ovulation [15].
In the present investigation, the progesterone level gradually decreased from day 0 (before starting protocol) to day 1 (before AI). This decreased level on day 1 might be due to the exogenous PGF2α injection which caused luteinisation of CL and physiological declination of progesterone towards the basal level for the manifestation of estrus [16].
The admirable conception rate in the modified select-synch protocol (Seld-synch) is attributed to the GnRH priming which sensitized the endocrine axis to obtain better follicular development and well synchrony between luteolysis, ovulation, and fixed-timed AI [1]. It causes ovulation of a mature follicle at the time of administration by inducing the LH surge. The last response observed select-synch might be due to the presence of a small less estrogenic dominant follicle [1] and might be due to divergence in the stage of the estrous cycle at the time of commencement of the protocol as well as the effect of hormonal doses and vitamin A injection [17]. The higher level of serum progesterone at the time of AI might be reminiscent of incomplete luteal regression following the administration of PGF2α which resulted in the least response in terms of conception rate in select-synch protocols of the present study [18]. The role of Vitamin A is also important in the development and maintenance of the pregnancy. Vitamin- A has a significant role in the reproductive outcome of the cow, both at the time of insemination and throughout pregnancy. Deficiency can even lead to either a complete failure of reproduction before implantation or fetal resorption or malformation [19].
Conclusion
There was much improvement in terms of oestrus response, serum mineral, and hormonal profiles along with conception in post-partum anestrus cows while using the modified select-synch protocol (Seld-synch). Finally, it can be concluded that the modified select-synch protocol (Seld-synch) is found to be a more effective method of the FTAI program. It can be recommended for the therapeutic management of post-partum anestrus cows provided that these animals do not have any uterine pathology or gross energy malnutrition.
