Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 56 issue 12 (december 2022) : 1468-1472

Vascular Perfusion of the Preovulatory Follicle and its Relationship in Pregnancy Establishment during Natural and Induced Estrus in Buffaloes

M. Selvaraju2,*, S. Prakash1, M. Palanisamy1, P. Visha1, R. Chitra1
1Department of Veterinary Gynaecology and Obstetrics, Veterinary College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Namakkal-637 002, Tamil Nadu, India.
2Veterinary University Training and Research Centre, Tamil Nadu Veterinary and Animal Sciences University, Karur-639 006, Tamil Nadu, India.
Cite article:- Selvaraju M., Prakash S., Palanisamy M., Visha P., Chitra R. (2022). Vascular Perfusion of the Preovulatory Follicle and its Relationship in Pregnancy Establishment during Natural and Induced Estrus in Buffaloes . Indian Journal of Animal Research. 56(12): 1468-1472. doi: 10.18805/IJAR.B-4277.

ABSTRACT

Background: In buffaloes, little attention has been devoted on the haemodynamics of preovulatory follicle (POF) and serum estradiol profiles and their relationship with conception rate following AI at natural and induced estrus. 

Methods: Sixty healthy pleuriparous graded Murrah buffaloes were selected and divided into two groups, viz., Group I and II. Group I buffaloes were artificially inseminated twice at an interval of 24 hours during natural estrus. Estrus induction was done using PIVD and PGF2α in group II buffaloes and AI was performed 48 h and 72 h after the withdrawal of PIVD. The blood flow of the preovulatory follicle (POF) and early pregnancy were assessed in both the groups by Doppler ultrasonography.

Result: On statistical analysis, the mean (± SE) of spectral indices revealed there was an increased blood flow to the POF during induced estrus (Group II) when compared to natural estrus (Group I). The conception rate observed was 66.67 and 58.33 per cent in group I and II, respectively. It is concluded that increased vascular perfusion to POF resulted in almost similar conception rates in induced and natural estrus following AI in buffaloes.

INTRODUCTION

Poor estrus detection and untimely insemination is the major cause for reproductive failures in buffaloes under field condition of India (Selvaraju et al., 2005). During the last 15 years, different hormonal treatment schedules for estrus synchronization and fixed time artificial insemination (AI) in buffalo have been proposed (Kang et al., 2007). Despite being widely studied, fertility in fixed time artificial insemination (FTAI) protocols still presents high variability (Colazo et al., 2004). Although, different types of synchronization programme are followed to avoid estrus detection methods and allow fixed time AI in buffaloes, varying rates of fertility rates were observed. Samir et al., (2019) correlated these variations in the fertility rates with vascular perfusion of preovulatory follicle (POF) and subsequent CL development in Egyptian buffaloes treated with CIDR-PGF2a and Ovsynch-CIDR.
       
Bovine follicles achieved ovulatory capacity at a diameter of approximately 10 mm (Sartori et al., 2001). A positive correlation was noticed between the follicle size at the time of AI (SaFilho et al., 2010) and corpus luteum size (Baruselli et al., 2003) after AI with pregnancy rate in cows. Varughese et al., (2014) observed that Doppler study proved to be better for accurate determination of quality of the follicle and to classify the CL as active or regressing in cows. Although, buffaloes are mainstay of the dairy industry in South and South East Asia, unlike the study in cows, little attention has been devoted in buffaloes on the haemodynamics of ovarian follicle and its relationship to serum steroid profiles and conception rate following AI at natural and induced estrus. Hence, the present study tested the hypothesis of a positive relationship of the vascular perfusion of the POF on successful establishment of pregnancy after estrus induction with Progesterone Impregnated Intra Vaginal Device (PIVD) and PGF2a in graded Murrah buffaloes.

MATERIALS AND METHODS

Current research study was conducted on 60 graded Murrah buffaloes at 60-70 days postpartum which were brought to the Artificial Insemination (AI) Unit of Veterinary Clinical Complex (VCC), Veterinary College and Research Institute, Namakkal, Tamil Nadu. The selected buffaloes were randomly and equally divided into two groups, viz., Group I (Control) and II (Treatment). Group I (Natural estrus group) buffaloes (n=30) were artificially inseminated twice (Double AI) at an interval of 24 hours during natural estrus. In group II (Induced estrus group) buffaloes (n=30), PIVD (TRIU-B®) were inserted with Progesterone Impregnated Intra Vaginal Device PIVD (TRIU-B®) on day 10 following natural estrus (Day of estrus was considered as day 0) and it was kept intra vaginally for 9 days. The PIVD was removed on 9th day after its insertion. One day prior to the removal of PIVD, 500 µg of PGF2a (2 ml, Cloprostenol) was administered intramuscularly and artificial insemination (AI) was performed 48 h and 72 h after the withdrawal of PIVD. Vascular perfusion of the POF was analysed during natural estrus in group I and natural and induced estrus in group II by Doppler ultrasonography. Early pregnancy diagnosis was done at day 28 after first AI using ultrasonography.
       
Buffaloes were restrained in the chute with caudal epidural anaesthesia by injecting 3 ml of 2% Lignocaine to minimize the movement of the animals during Doppler ultrasonic imaging. A real time B mode and color Doppler ultrasound scanner (SONORAY) equipped with liner array, 7.5 MHz frequency transrectal transducer was used for transrectal scanning. The power-Doppler mode was used to display blood-flow signals from the vessels of the follicle wall. Gray-scale and Doppler end points were evaluated while the entire follicle was being assessed in a slow continuous motion several times as described for mares Silva et al., (2006). For spectral Doppler assessment, the sample cursor or gate was set at a width of 1 mm in the most prominent color signal in the vessel of the POF wall. The angle of the ultrasound beam in relation to the direction of blood flow in the vessel (i.e. insonation angle) was unknown and therefore, relative velocities for group comparisons, rather than actual velocities, were obtained (Ginther, 2007). Doppler spectrums of three cardiac cycles were generated and one of the cycles was used for spectral measurements. The mean value of two measurements for each end point was used in the statistical analysis. The velocity end points from spectral Doppler ultrasonography of a vessel in the wall of the POF were peak systolic velocity (PSV), time-averaged maximum velocity (TAMV) and end diastolic velocity (EDV). The follicle blood perfusion end points were pulsatility index (PI) and resistance index (RI) and were computed from the velocity measurements by the scanner. Doppler measurement techniques and other aspects of blood velocities and perfusion indices have been described as per Zwiebel and Pellerito (2005) and Ginther (2007). The paired and unpaired T-test method was followed for the experiment (Snedecor and Cochran, 1994) and the data collected were analyzed using SPSS® 20.0. Software package. The data related to the conception rate was analyzed by the chi-square test.

RESULTS AND DISCUSSION

POF blood flow
 
The spectral indices viz., PI, RI and TAMV of the POF during natural and induced estrus in cyclical postpartum Murrah buffaloes are depicted in the Table 1. On statistical analysis, at the time of natural estrus in group I and natural and induced estrus in group II, there was a high significant (P<0.01) difference in all the mean (± SE) spectral indices (PI, RI and TAMV) between pregnant and non-pregnant buffaloes. The mean (± SE) PI and TAMV in pregnant or non-pregnant buffaloes in group I and II natural estrus significantly differed from the mean (± SE) PI and TAMV of pregnant or non-pregnant buffaloes of group II induced estrus. Although the mean (± SE) RI value decreased in pregnant and non-pregnant buffaloes in induced estrus in group II when compared to natural estrus group I and the differences were not statistically significant. But significant and non-significant reduction in mean (± SE) RI value in induced estrus of group II was found when comparing the natural estrus of same group in pregnant and non-pregnant buffaloes, respectively. The overall mean (± SE) PI and TAMV of induced estrus (group II) significantly differed from natural estrus of group I and II. Although there was a reduction in mean (± SE) RI value in induced estrus of group II when compared to natural estrus of group I, the difference was not statistically significant. But significant difference in overall mean (± SE) RI was found between natural and induced estrus in group II. The present study indicates, the POF of group II buffaloes (induced estrus) had statistically significant decrease in PI and increase in TAMV when compared to the group I buffaloes (natural estrus). This finding of our study clearly proved that there was an increased blood flow to the wall of the POF in group II buffaloes (induced estrus) (Fig 1) when compared to the group I buffaloes (natural estrus) (Fig 2). Similar finding was reported by Ginther et al., (2014) in heifers. The greater blood flow in the follicle is related to expression of more concentration of vascular endothelial growth factor (VEGF) and greater dissolved oxygen in the follicular fluid (Samir et al., 2019). A greater increase in blood flow to the POF of group II buffaloes indicated an increased blood circulation from an increase in number and diameter of the arterioles that form a network encasing the follicle (Hunter, 2003). An increase in number and diameter of arterioles agrees with decreased RI and PI and increase in TAMV in the group II buffaloes. The pre-ovulatory follicle theca layer develops a thin capillary tissue over the granulosa layer and the increase in blood flow into the theca layer enhances the follicular development (Yamada et al., 1994). This might be the reason for increased circulating estradiol levels in the group II buffaloes (Table 3). Estradiol has been supposed to be one of the important factors in the regulation of vascular perfusion in the reproductive tract due to its vasodilatory function (Samir et al., 2019).
 

Table 1: Haemodynamic analysis of POF of natural and induced estrus in buffaloes.


 

Fig 1: Increased blood flow to the POF during the induced estrus


 

Fig 2: The blood flow to the POF during the natural estrus.


 
In the selection of the POF, the VEGF and Fibroblastic growth factor (FGF) families were involved in the proliferation of capillaries that resulting in an increased supply of nutrients and precursors to the POF and therefore supporting the growth of its dimension (Berisha et al., 2000). Mitochondria play a vital role in the oocyte in providing ATP for activation at the time of fertilization and for pre-implantation embryo development. The reduced blood flow to the POF in buffaloes that did not become pregnant in present study might be due to reduced mitochondrial oxidative phosphorylation in the oocyte as explained by Siddiqui et al., (2008).
 
Ovulation and conception rate
 
Ovulatory response and conception rate following natural estrus and estrus induction with PIVD and PGF2α in buffaloes is presented in Table 2. The percentage of ovulatory response was 100.00 per cent in group I and II and hence, no difference was noticed in ovulatory response between them. The 100 per cent ovulatory response in the present study might be due to the greater angiogenesis and increased secretion of estradiol 17 β in the induced estrus buffaloes as explained by De Tarso et al., (2016).
 

Table 2: Ovulatory response and Conception rate following natural estrus and PIVD + PGF2á treatment in buffaloes.


       
The conception rate observed was 66.67 and 60.00 per cent in group I and II, respectively. The overall conception rate recorded in cyclical postpartum Murrah was 62.50 per cent. In the present investigation, the conception rate obtained following induced estrus was almost similar to that of natural estrus. Hence, it indicated that PIVD + PGF2α found to be effective in terms of increasing the vascular perfusion of POF for obtaining good fertility rate in buffaloes as explained by De Tarso et al., (2016).
 
Estradiol concentration
 
The mean serum estradiol 17β (pg/ml) level of buffaloes during natural and induced estrus are presented in Table 3. In group I, although the mean (± SE) serum estradiol 17 β level was greater in pregnant animals when compared to the non-pregnant animals, statistically the difference was not significant (P>0.05) at estrus. During induced estrus in group II, a significant (P<0.01) difference in mean (± SE) serum estradiol-17β (pg/ml) level between pregnant and non-pregnant buffaloes was found. The mean (± SE) serum estradiol-17β (pg/ml) level of group I pregnant buffaloes during natural estrus differed significantly (P<0.01) from group II pregnant buffaloes during induced estrus. However, no significant (P>0.05) difference was observed between non-pregnant buffaloes during natural estrus in group I and induced estrus in group II.
 

Table 3: Mean (±SE) serum estradiol-17â (pg/ml) levels during natural and induced estrus in buffaloes.


       
The elevated level of estradiol 17β at the time of estrus might be a reason for better conception in buffaloes of both groups. The alteration in estradiol and progesterone ratio by the increased level of estradiol during estrus might favour the fertility in the buffaloes of our experiment as explained by Lopes et al., (2007). The occurrence of high serum estradiol concentration on the day of estrus in pregnant buffaloes than non-pregnant buffaloes of this study suggested that steroid biosynthesis by the POF influenced pregnancy establishment (Lopes et al., 2007). The mean serum estradiol 17β level during induced estrus in both pregnant and non-pregnant buffaloes was higher than the pregnant and non-pregnant buffaloes of natural estrus of group I. It indicated that estrus induction program with PIVD + PGF2α caused the emergence of a new follicular wave with larger POF which in turn secreted more amount of estradiol at the time of induced estrus. The increased estradiol concentration at induced estrus might be the reason for almost similar pregnancy rates in natural and induced estrus buffaloes as described by Quirk et al., (2004).

CONCLUSION

Pregnancy establishment in buffaloes has been reported to be influenced by the blood flow to the POF during estrus. Based on the vascular perfusion of the POF and serum estradiol 17β concentration, it is concluded that the conception rate obtained following induced estrus was almost similar to that of natural estrus. Hence, it is concluded that PIVD + PGF2α found to be effective to increase the vascular perfusion of POF during induced estrus for obtaining good fertility rate as that of natural estrus without the necessity of estrus detection in buffaloes.

ACKNOWLEDGEMENT

We thank the Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai for all the facilities provided to conduct this work.

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