Timed Artificial Insemination Methods involving Ovulation of Second Follicular Wave vis-à-vis Extension in Growth of Dominant Follicle- New Alternatives to Enhance Fertility in Buffalo (Bubalus bubalis)

The study was undertaken to assess the ovarian and fertility responses following ovulation of dominant follicle (DF) from second follicular wave (2nd FW) through application of modified synchronization protocols in 46 cyclic postpartum buffaloes. All buffaloes were administered PGF2α (PGF; Cloprostenol, 500 mcg) on day -2, gonadotrophin releasing hormone (GnRH; Buserelin) on days 0 (10 mcg) and 7 (20 mcg). After that, all buffaloes were randomly divided into 2 groups on the basis of administration of second dose of PGF (500 mcg) on day 14 (Group I, n= 20) and 15 (Group II, n= 26). Third dose of GnRH (10 mcg) was administered on day 16 (Evening) and 17 (Morning) in group I and II, respectively, followed by artificial insemination (AI) at 12-16 h later. The pregnancy diagnosis was performed at day 45 post AI. Progesterone (P4) concentrations remained higher during 2FW in buffaloes that became pregnant compared to nonpregnant ones. A larger (P<0.01) diameter of the DF was observed on day of third GnRH and AI in group II compared to group I buffaloes. A comparable corpus luteum (CL) diameter was observed on all days of protocols in both groups. Ovulatory response following last GnRH administration and first AI pregnancy rate were 90 vs. 96 % and 35 vs. 46 % in group I vs. II, respectively. In conclusion, ovarian and fertility responses following ovulation of DF from 2nd FW showed an improved trend in buffaloes where growth of DF was extended by delaying the luteolysis.

The buffalo is the backbone of dairy industry in India contributing more than 55 per cent of national total milk production. Reproductive efficiency is the primary factor affecting productivity of buffalo. Low reproductive efficiencies due to problems of subestrus, anestrus, and repeat breeding are considered as major constraints in buffalo farming (Dhindsa et al., 2016). In fact, decline in conception rate and increase in calving interval due to AI at wrong time is a major problem in the progress of buffalo dairy industry. Applying estrus synchronization treatments in buffaloes provide a potential alternative for increasing their productive period as these regimens eliminate the problem of estrus detection. Even though a number of protocols have been implicated, a partial success has been achieved with available estrus synchronization regimens till date in buffaloes (Ghuman et al., 2010).
Moreover, most of the protocols involve timed AI (TAI) following ovulation of the DF from first follicular wave (1 st FW). However, the DF of the 1 st FW and 2 nd FW grows in different P 4 environments. Various studies have demonstrated favourable effect of P 4 environment during DF development on subsequent CL development, CL functionality, estrus behaviour and fertility in bovine (Lonergan, 2011;Wiltbank, 2012;Dadarwal et al., 2013;Bilal et al., 2017). Studies in cattle (Denicol et al., 2012) have shown that fertility decreases when 1 st FW DF is allowed to ovulate than the 2 nd FW. Thus the present study was conducted to suggest modified estrus synchronization protocols involving ovulation of 2 nd FW DF and to ascertain the effect of delaying luteolysis during the treatment on ovarian response and hormonal profile of buffaloes.

Animals
The present study was carried out on 46 post-partum (> 60 days in milk) cyclic buffaloes maintained at dairy farm, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. The selected buffaloes had body condition score (BCS) ranging from 2.5 to 3.5 on scale of 1 to 5 basis (Edmondson et al., 1989). The animals were managed in a semi-intensive housing system. Only buffaloes in general good health and free from genital abnormalities / discharges were included in the present study. The cyclic status of all buffaloes was assessed by transrectal ultrasonographic examination of ovarian structures at the start of the experiment.

Experimental design
All buffaloes were pre-synchronized at random stages of the estrous cycle by administering single injection of PGF2α (PGF: Cloprostenol B.P 500 µg, im, Provimi Vetcare) on -2 day. Two days later (day 0), all animals were administered a gonadotrophin releasing hormone analogue (GnRH: Buserelin acetate 10 mcg, im, MSD) to induce ovulation and a new FW. Again on day 7 all animals were given GnRH (20 mcg) to ovulate DF of 1 st FW and initiate 2 nd FW. Thereafter to study the effect of increased P 4 exposure on the growth of 2 nd FW DF, all buffaloes were randomly divided into 2 groups (Group I, n=20; Group II, n=26) on the basis of administration of second dose of PGF (500 µg) that was injected one day late in group II compared to group I buffaloes (15 vs. 14 day). A third dose of GnRH (10 mcg) was administered on day 16 (Evening) and 17 (Morning) in group I and II, respectively, followed by AI at 12-16 h later.

Blood collection and transrectal ultrasonography
All buffaloes of both groups were subjected to blood sampling and transrectal ultrasonographic examination of ovarian structures on respective days of treatments as shown in fig. 1. Blood samples (5-10 ml, jugular venupuncture) were collected into heparinised (1:1000) tubes which were immediately placed in ice box and transferred to the laboratory. Plasma was separated by centrifugation (3000 rpm, 15 minutes) and stored at -20°C until the hormone assay. Transrectal ultrasonographic examination was performed using brightness mode (B mode) ultrasound device, Z 5 diagnostic ultrasound machine (Shenzhen Mindray Biomedical Electronic co. ltd). The animals were restrained properly to avoid distortion in the ultrasound image. During each ultrasonographic examination the number and diameter of DFs and CLs were measured after freezing the best image with minimum distortion and maximum clarity.

Progesterone analysis
Plasma P 4 was estimated by liquid phase Radioimmunoassay (RIA) procedure using progesterone antisera raised in the department of Veterinary Gynaecology and Obstetrics, GADVASU, Ludhiana (Ghuman et al., 2009). The sensitivity of the assay was 0.1ng/ml for P 4 . The mean intra-and inter-assay coefficients of variance were 6.0 and 9.3 percent, respectively.

Statistical analysis
The data were presented as mean ± standard error of mean. Chi square test was applied to compare ovulation and pregnancy rates. Plasma P 4 , DF and CL values were analysed using "Unpaired T-test" to compare between groups and "One way ANOVA" to evaluate the effect within the group. The statistical significance of tests was determined at P<0.05 and P<0.01 levels.

Ethical approval
This study was approved by the Institutional Animal Ethics Committee, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana.

RESULTS AND DISCUSSION
Exposure to insufficient concentrations of P 4 during the growth of the ovulatory follicle is one of the important factors that affect fertility in high-producing dairy animals (Wiltbank et al., 2012;Bisinotto et al., 2010). Low P 4 concentration during the development of DF reduces the subsequent fertility in bovine (Wiltbank et al., 2012). The DF from 1 st FW develops under low circulatory P 4 whereas DF of 2 nd FW/ovulatory wave grows under luteal level (> 2 ng/ml) of P 4 , prior to luteolysis (Denicol et al., 2012). In present study, P 4 concentrations on days of second PGF, third GnRH and AI were 1.84±0.17, 0.54±0.02 and 0.09±0.05 ng/ml in group I, while the corresponding levels in group II were 1.96±0.18, 0.44±0.02 and 0.16±0.08 ng/ ml, respectively. The present results support the hypothesis that higher P 4 levels are required during the growth of ovulatory follicle to enhance pregnancy rate. In group I, P 4 concentrations during 2 nd FW on days of PGF, GnRH and AI in pregnant vs. non-pregnant buffaloes were 2. The mean DF and CL size on various days of modified synchronization protocols in group I and II is presented in Table 1. The DF on the day of third GnRH and AI was significantly (P<0.01) larger in group II compared to group I buffaloes (13.06±0.25 and 14.47±0.26 vs. 11.53±0.20 and 11.92±0.14 mm, respectively). The size of the CL did not differ significantly (P>0.05) between the groups. This finding supported the earlier report suggesting uncertain correlation between size of the CL determined by conventional ultrasonography and P 4 secretion or subsequent pregnancy (Russo et al., 2010).
In group I and II, 90 and 96 percent buffaloes ovulated following last GnRH injection, respectively. The observed ovulatory responses during second wave in buffaloes were higher than in earlier studies (78.1%: Souza et al., 2008;83.3%: Hoque et al., 2014). The overall first AI pregnancy rate was 35 and 46 percent in group I and II, respectively. Earlier research has revealed that increasing P 4 during growth of the follicular wave increased fertility more than 10 percent to the subsequent AI in bovines (Bisinotto et al., 2010). During the current study, a higher pregnancy rate by 11 per cent was observed in group II (46%) compared to group I (35 %). The pregnancy rate obtained in the present study is in corroboration with earlier studies comparing conception rate following second wave ovulated AI (Cunha et al., 2008;Hoque et al., 2014). The exact mechanisms that produce increase in fertility are unknown. However, the possible underlying mechanism by which P 4 modulates fertility likely involves a change in the pattern of LH release (Wiltbank et al., 2012). Changes in LH pulse frequency are associated with alterations in the process of follicular maturation and subsequent embryo survival (Cerri et al., 2011). In buffaloes only a few reports are available supporting the above hypothesis  (Hoque et al., 2014). Moreover, no literature is available to compare the present results of extending growth of DF under high P 4 environment during 2 nd FW on subsequent fertility in buffaloes. Future studies are required in this direction to define the mechanism involved and support the hypothesis.

CONCLUSION
The results of the present study indicate that TAI protocols involving ovulation of the 2 nd FW may successfully be incorporated to enhance the reproductive efficiency of buffaloes. The delay in luteolysis during 2 nd FW may increase the preovulatory DF size by extending its growth under high P 4 environment that subsequently improves the oocyte quality and pregnancy rate in buffaloes.