Propuesta para la disminución de los requisitos en los trámites

The percentage of heifers that ovulated following treatment in the artificial insemination trial was 80% (36/45). Two heifers were not inseminated because it was not possible to reach the uterine lumen in order to deposit the semen. From the total number of heifers inseminated (n=43), three were confirmed pregnant 35 days post-AI.

9.5 Discussion

Studies of the effect of different letrozole treatment regimens on ovarian function in cattle have provided evidence of the potential of aromatase inhibitors as a tool to manipulate ovarian function in this species ([39, 40] Chapter 5, 6 and 8). The data presented herein further support

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this notion by confirming our hypothesis that extended letrozole treatment using intravaginal devices, combined with a single PGF and GnRH treatment, increases the percentage of heifers that ovulated and the synchrony of ovulation, regardless the stage of the estrous cycle at initiation of the protocol. However, letrozole-PGF-GnRH-based protocol for ovulation synchronization resulted in poor pregnancy rates

The percentage of heifers that ovulated was increased by the addition of a 4-day regimen of letrozole to a PGF plus GnRH protocol as compared to PGF plus GnRH alone (87.1% vs 69.4%, respectively). This increase in ovulatory response and synchrony of ovulation after the addition of letrozole may involve more follicles responding to the GnRH treatment and a decrease in early ovulations (ovulations that occurred prior to GnRH treatment). We have reported previously that letrozole induced growth and prolonged the lifespan of dominant follicles by increasing circulating plasma LH concentrations ([39, 40] Chapters 5 and 6). We speculated that the addition of letrozole to the PGF plus GnRH protocol would allow for smaller, less competent follicles, which otherwise would not have responded to GnRH treatment, to reach the necessary diameter and LH receptor populations to acquire ovulatory capacity and ovulate within 48 h post GnRH treatment. In addition to promoting follicular growth, letrozole treatment likely prolonged the lifespan of the static dominant follicles that otherwise would have become atretic by the time of GnRH treatment ([39, 40], Chapter 8). The reduction in early ovulations is supported by the observation that letrozole prevents estradiol secretion (Chapters 5, 6 and 8) thus minimizing the occurrence of a pre-ovulatory rise in estradiol concentration and a LH surge prior to administration of GnRH. The improved ovulatory response and synchrony of ovulation after GnRH indicated that letrozole could be applied for the development of a FTAI protocol.

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Pregnancy rates were low in the small breeding trial; only 3 of 43 inseminated heifers were pregnant 35 days post-AI. The effect of letrozole treatment on fertility was unexpected. We have reported that letrozole treatment affects the plasma hormone profile and steroidogenesis, thus it is also expected to alter other aspects of reproductive function such as the duration of follicle dominance, oocyte age and activation, the process of fertilization, embryo quality, and CL lifespan. However, it is possible that other factors unrelated with the non-steroidal aromatase inhibitor treatment may also have influenced the pregnancy outcome obtained in this study.

The impact of duration of dominance of the pre-ovulatory follicle on timing of ovulation and fertility has been reported [264, 265]. Prolonged dominance of the ovulatory follicle has been associated with reduced pregnancy rates. The decrease in fertility was more profound after 9 days of dominance (35 to 70% reduction in pregnancy rates) compared to after 2 days of dominance [264]. Duration of dominance is related to increased LH levels [266] and early activation of oocytes (resumption of meiosis) has been associated with decreased fertility [264, 266-268].

During the present study, we attempted to design a minimal duration of follicle dominance in the pre-ovulatory follicles by adjusting the length of letrozole treatment to 4 days. The duration of dominance of the pre-ovulatory follicles among groups was estimated based on the average day of emergence of the follicle that became the ovulatory follicle - follicles typically reach dominant status by 3 days post-wave emergence (2.8 days) [65]. It would follow that the duration of dominance in Day 0 and 4 groups were 4 and 8 days, respectively. Day 8 group contained heifers that would have had follicles from the first follicular wave (n=4) and heifers with follicles originating from the second follicular wave (n=4). Hence, heifers in this group

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would have had follicles in which dominance would have been 4 days or 12 days at the time of ovulation. Day 12 group should have had follicles that originated from only the second wave, and would have been dominant for about 4 days before ovulation. Day 16 group would have had follicles originating from either the second or third wave of follicular growth and the duration of dominance in this group would have been 4 and 12 days (4 and 6 heifers, respectively). As evidenced by these numbers, heifers between Days 7 and 9 (Day 8 ± 1) and between Days 15 and 17 (Day 16 ± 1; Day 0 = ovulation) would be at risk of developing an ovulatory follicle that had been dominant for approximately 12 days and would likely have an aged and/or prematurely activated oocyte. However, we must consider the possibility that increased LH secretion (caused by letrozole treatment [39, 40]) may also cause premature activation of oocytes and a reduction in fertility, even if duration of dominance of the pre-ovulatory follicle was within normal range (1 to 5 days, [268]).

Estradiol concentrations were reduced in heifers in Groups 0 and 4, tended to be reduced in Group 8, while Group 12 did not differ from their respective controls. Considering that estradiol concentration at device removal did not differ among letrozole-treated groups and averaged 2.1 ± 0.24 pg/mL, the differences noted between letrozole-treated heifers relative to their controls were attributed to changes in estradiol concentration in the control samples. Estradiol concentration in the control heifers Days 4, 8, 12 and 16 were consistent with those obtained previously: basal estradiol concentrations have been reported to be around 2 pg/mL, with a small rise between Days 4 and 7 post-LH peak and no significant changes in estradiol concentration thereafter until the next pre-ovulatory estradiol rise [55]. It is possible, however, that the presence of a newly recruited wave of follicular development (third wave) was responsible for the low estradiol

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concentration observed in the control heifers on Day 16. In other words, the high estradiol concentration expected with the presence of a growing (estrogen-active) follicle from the second wave (potentially an ovulatory wave) may have been obscured by the coexistence of atretic (estrogen-inactive) follicles from the second follicular wave with newly recruited follicles which have not yet reach their maximal estrogen production potential.

There is some controversy regarding the requirement of estradiol for final follicular maturation and the presence of fertilizable oocytes in mammals. In rhesus monkeys, aromatase inhibitor treatment during the late follicular phase did not alter the number nor the pattern of growth of follicles although oocyte activation and in vitro fertility was reduced [139]. While some studies reported that addition of estradiol in in vitro maturation protocols impaired bovine oocyte nuclear maturation and subsequent embryo development [269, 270], others found that estradiol was essential for normal in vitro maturation [271], especially of early antral follicle- derived oocytes [272]. However, data on the effects of estradiol deprivation on bovine oocyte maturation in vivo are not available, most likely due to the lack of an efficient treatment regimen to mimic such a condition. Although not directly assessed in this study, we can presume that follicular environment has been affected by treatment, affecting oocyte quality by disturbing meiosis. However, treatment with anastrozole, another non-steroidal aromatase inhibitor, did not impair follicular growth, ovulation nor fertilization in vivo and embryo development in vitro using a mouse model [273]. In addition, there are several important extra-gonadal effects of the estradiol produced by growing pre-ovulatory follicles, such as the development of receptive endometrium, the production of the cervical mucus, and sperm transport [274]. All these processes could be negatively affected by letrozole-induced estradiol inhibition. This notion is

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further supported by the observation that heifers showed normal signs of estrus after initial PGF and GnRH treatment (control ovulations) but did not exhibit any estrous behaviour (i.e., mounting, standing to be mounted, vaginal mucus) following PGF and GnRH treatment after letrozole treatment.

The effect of letrozole treatment on CL lifespan in this study was also unexpected. In previous studies, letrozole treatment resulted in larger CL which secreted higher levels of progesterone [39, 40] (Chapters 5 and 6). Thirty out of 48 (62.5%) heifers treated had CL considered to be of normal diameter at last observation (9 days post-letrozole treatment ovulation), while 18 (37.5%) heifers underwent luteolysis prior to the last observation at 9 days after ovulation. Progesterone production was not affected by group and its profile corresponded to CL lifespan (i.e., normal vs short lifespan). The reason for these differences on CL lifespan within groups remains unclear. There appeared to be no relationship between duration of follicular dominance and lifespan of the resulting CL. Short-lived CL have been described following hCG-induced ovulation of the dominant follicle of the first follicular wave in cattle, suggesting that pre-ovulatory changes intrinsic to the treatment may be responsible for the abnormal CL function [275].

Another possible explanation for the observed short lifespan CL is related with the occurrence of early luteolysis. Short luteal phases in 33% of cows [276] and 47 % of heifers have been reported [277] when GnRH treatment was given 24 h after PGF. The short luteal phases were related to early release of PGF2α from the endometrium [278]. Reduced estradiol concentration during the proestrus has also been linked to short luteal lifespan. It has been hypothesized that high estradiol concentrations during proestrus are needed in order to induce an

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adequate number of progesterone receptors, thus allowing progesterone to regulate the uterine secretion of PGF [263, 279]. Therefore, low estradiol concentration induced by letrozole treatment may impair the inhibitory effect that progesterone has on PGF secretion by allowing the increase in number of estrogen and oxytocin receptors in the endometrium and early release of PGF2α [280].

An important limitation of the present AI trial is the lack of a letrozole-free control group. Therefore, it is difficult to determine the impact that factors such as AI technician, semen quality and semen handling had on pregnancy rates. Another important factor to consider is the timing of the inseminations. Heifers were inseminated 24 h after GnRH based on an earlier study in which it was reported that ovulations occurred between 24 and 32 h after GnRH treatment [94, 98]. However, it is unknown if letrozole treatment alters the window of time between GnRH treatment and ovulation. Daily ultrasound examinations did not allow the determination of the time of ovulation precisely in the present study. Finally, the presence of the short lifespan CL during the AI trial also needs to be considered, which would reduce the proportion of heifers that could have remained pregnant. Post-AI ultrasound examinations were not performed until pregnancy check; hence, no information on CL lifespan is available for this set of animals.

In summary, the addition of a letrozole-impregnated intravaginal device for 4 days, combined with PGF treatment at device removal and GnRH 24 h post-device removal increased the percentage of ovulations and synchrony of ovulation in cattle, regardless the stage of the estrous cycle at initiation of treatment. Reduced luteal lifespan after letrozole treatment was unexpected and requires further investigation in order to elucidate the mechanism responsible for this observation. Although the results obtained in the AI trial are unfavorable, we consider that

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this portion of the study needs to be repeated with the inclusion of control group and in vitro fertilization trials in order to draw more meaningful conclusions on the impact that letrozole treatment has on oocyte competence and fertility. Adjustments of timing of AI, interval from PGF to GnRH treatment and even in vitro assessment of oocyte fertizability after estradiol deprivation may be considered. Finally, the letrozole treatment regimen presented herein has potential as a model to investigate the effect of estradiol deprivation on oocyte maturation and fertility in vivo.

We conclude that the addition of letrozole to a GnRH plus PGF protocol can be used to increase the number of animals ovulating and the synchrony of ovulation, but additional studies are needed in order to elucidate the mechanisms related to reduced fertility observed herein.