Analysis of factors affecting embryo recovery in superovulated buffaloes

Field: Veterinary (Health Sciences)

The main objective of this thesis was to investigate the different factors affecting the embryo recovery in superovulated buffaloes. Several hypotheses have been formulated over the years to explain the low embryo recovery recorded in this species after superovulatory treatment, among which the most likely is a failure of ovum capture. Before addressing the major issue, other experiments were carried out in order to select both the donors and the most suitable superstimulation treatment. Indeed, another critical factor limiting the application of this technology in buffalo is the high between-animal variability in the number of embryos produced. Therefore, the aim of Experiment 1 was to evaluate whether the Anti-Mullerian hormone (AMH) can be used as a reliable marker to select good donors in addition to the screening of the antral follicular count (AFC), before enrolling buffaloes in embryo production programs. In order to do so, 54 multiparous Italian Mediterranean Buffalo cows were synchronized by double prostaglandin administration spaced 11 days apart and were slaughtered at a local authorized abattoir 6 days after the second administration. On all animals AFC, recovery rate and oocyte quality were recorded, and both plasma and follicular fluid (FF) were collected for AMH determination. In a representative number (n=22) the mRNA expression of six genes (AMHR2, CYP19A1, FSHR, LHR, TP53INP1, and CASP3) was analysed in granulosa cells (GCs). Finally, buffaloes were classified according to the AFC as good (≥ 12 follicles) and bad (< 12 follicles) donors, in order to evaluate the differences in reproductive parameters. Differences in AMH concentration in FF and gene expression in GCs among classes of follicles were analysed by ANOVA. Pearson correlation analysis was carried out to evaluate the relationship between AMH and the AFC, as well as among other reproductive parameters. The differences in AMH plasma levels and reproductive parameters between good and bad donors were analysed by Student’s t test. The results of Experiment 1 showed a positive correlation (r = 0.31; P<0.05) between intrafollicular AMH concentration and the AFC in buffalo. Interestingly, good donors had a higher (P < 0.05) concentration of AMH in FF and AMHR2 levels in small follicles and higher (P < 0.05) LHR levels in large follicles than bad donors. However, no correlation was found with the plasmatic AMH levels, likely due to the poor sensitivity of the available kits. Thus, one of the future objectives could be to assess a more reliable specific assay in order to use this as a marker to screen donors. The aim of Experiment 2 was to evaluate the efficacy of replacing the last four FSH injections with a single administration of 1000 IU of eCG compared to the conventional FSH decreasing doses superovulation protocol. The study was carried out on 20 multiparous Italian Mediterranean buffaloes synchronized with a common double prostaglandin protocol. Then, animals were divided in two groups: one treated with a superovulatory protocol with FSH decreasing doses and one with the eCG replacing the last four FSH administrations. All cows were artificially inseminated at oestrus and ultrasonographic investigations in the two groups were carried out to assess follicular dynamics and to establish the number and size of corpora lutea on the ovaries. Differences between treatments were analysed by Student’s t test. It was demonstrated that the replacement of the last four injections of FSH with a single administration of eCG within a superovulation protocol in buffalo is not recommended, due to the presence of a high number of anovulatory follicles at the moment of flushing, and a reduced number of ova compared to the conventional FSH treatment (3.0 ± 0.6 vs 6.6 ± 0.9 and 2.0 ± 1.2 vs 0.8 ± 0.6 respectively). However, a poor recovery rate in relation to the number of corpus luteum (CL) recorded in both protocols remains. In order to select the most reliable control group for the superovulation study, the aim of Experiment 3 was to evaluate the relationship between the ovarian follicular response at the start of an Ovsynch program and the pregnancy outcome. The study involved 116 pluriparous buffaloes that were synchronized by Ovsynch–TAI program, involving the administration of GnRH on Day 0 (GnRH1), PGF2a on Day 7, and GnRH on Day 9 (GnRH2), with timed artificial insemination (TAI) carried out on Day 10. Ovarian ultrasound examinations were undertaken on days 0, 2, 7, 9, 10, and 11 to record total follicles, the dimensions of follicles greater than 0.75 cm, dimensions of the CL, and the occurrence of ovulation. Blood concentrations of progesterone (P4) were measured on days 0 and 10 and 10 days after TAI. Pregnancy status was determined on days 27 and 45 after TAI to calculate late embryonic mortality. The data were analysed by ANOVA and multiple logistic regression. The results of Experiment 3 showed that only 60% of the buffaloes ovulated after the first GnRH on day 0 of the Ovsynch-TAI program. A higher proportion (P <0.01) of buffaloes that ovulated after GnRH1 had a vascularized corpus luteum (CL) on day 7. Furthermore, a greater proportion (P<0.05) of buffaloes that ovulated after GnRH1 was in oestrus and both a higher ovulation rate and pregnancy to AI were observed compared to buffaloes that did not ovulate. The results of the experiment 3 showed how important is the ovulation after the 1st GnRH to the pregnancy outcome, during Ovsynch-TAI program. This is due to both an optimized response of the CL to PGF2α on day 7 and a better response of the preovulatory follicle to second GnRH on day 9, increasing the likelihood of pregnancy to TAI. The results of this experiment allowed us to better plan the experiment 4, by selecting the animals that ovulated after the 1st GnRH as a control group to compare with superovulated animals. Therefore, the aim of Experiment 4 was to compare the morphological parameters of the follicles and the steroid profile (progesterone and oestradiol) both in plasma and follicular fluid in the peri-ovulatory period in superovulated vs synchronized buffaloes. Thirty-five multiparous Italian Mediterranean Buffalo cows were randomLy divided into two groups: a group (n = 25) was synchronized by Ovsynch and another group (n=10) was superovulated with conventional FSH protocol and all of them were sacrificed 18 h after GnRH. On all animals AFC, recovery rate and oocyte quality were recorded, and both plasma and FF were collected for steroid profile determination. In addition, out of 10 animals (5/group), GCs were collected to analyse the mRNA expression of gonadotropin receptors LHR and FSHR while oviducts were collected to evaluate the mRNA expression of steroid receptors ER1 and PGR, VEGF and the VEGF receptor FLK1. Differences in morphological follicular parameters, as well as in the concentration of progesterone and oestradiol both in plasma and in follicular fluid were analysed by ANOVA, while differences in the recovery rate and in the percentage of oocytes showing cumulus expansion were analysed by Chi Square Test. Experiment 4 demonstrated that in superovulated animals the E2 and P4 concentrations in FF were lower (P<0.05) than in synchronized animals. Interestingly, both the recovery rate and the percentage of oocytes exhibiting proper cumulus expansion decreased (P<0.05) in the superovulated animals. Finally, in superovulated buffaloes a decreased expression of both ER1 and the VEGF receptor FLK1 in the infundibulum and an increased expression of FSHR in granulosa cells were observed (P<0.05). The results of the latter experiment suggest that the exogenous FSH treatment has probably increased the expression of FSHR in granulosa cells that was not followed by a parallel increase of oestradiol synthesis, eliciting an alteration of the granulosa cell function. The consequent altered steroid profile could affect in turn both the cumulus cells expansion during maturation, and the contraction-relaxation of the infundibulum, confirming the hypothesis of a failure in ovum capture in superovulated buffaloes. In conclusion, the results of this thesis taken together highlight the limitations of MOET in buffalo, suggesting that at present the only way to improve the intensity of genetic selection, through the maternal lineage, in this species is the OPU-IVEP technology. Indeed, buffaloes do respond to the gonadotropin treatment with multiple ovulations but the ova capture is severely impaired, resulting in poor embryo recovery. Nevertheless, the outputs of this thesis led to an improved knowledge and understanding of the limiting factors of the superovulation technique in this species.